Glial cell line-derived neurotrophic factor receptor

ABSTRACT

The present invention relates to glial cell line-derived neurotrophic factor (GDNF), a potent neurotrophin that exhibits a broad spectrum of biological activities on a variety of cell types from both the central and peripheral nervous systems. The present invention involves the cloning and characterization of a high affinity receptor for GDNF. This molecule has been named GDNF receptor (GDNFR) since it is the first known component of a receptor system. Nucleic acid and amino acid sequences are described for GDNFR protein products. A hydrophobic domain with the features of a signal peptide is found at the amino terminus, while a second hydrophobic domain at the carboxy terminus is involved in the linkage of the receptor to the cell membrane. The lack of a transmembrane domain and cytoplasmic region indicates that GDNFR requires one or more accessory molecules in order to mediate transmembrane signaling. GDNFR mRNA is widely distributed in both nervous system and non-neural tissues, consistent with the similar distribution found for GDNF.

[0001] This application is a continuation of U.S. application Ser. No.08/837,199, filed Apr. 14, 1997, now pending, which claims the benefitof U.S. provisional application serial No. 60/015,907, filed April 22,1996 and U.S. provisional application serial No. 60/017,221, filed May9, 1996, which are incorporated by reference herein.

1. FIELD OF THE INVENTION

[0002] The present invention relates to receptors for glial cellline-derived neurotrophic factor (GDNF) and provides nucleic acid andamino acid sequences encoding GDNF receptor (GDNFR). The presentinvention also relates to therapeutic techniques for the treatment ofGDNF-responsive conditions.

2. BACKGROUND OF THE INVENTION

[0003] Glial Cell Line-Derived Neurotrophic Factor

[0004] Glial cell line-derived neurotrophic factor (GDNF) was initiallyisolated and cloned from rat B49 cells as a potent neurotrophic factorthat enhances survival of midbrain dopaminergic neurons (Lin et al.,Science, 260, 1130-1132, 1993). Recent studies have indicated that thismolecule exhibits a variety of other biological activities, havingeffects on several types of neurons from both the central and peripheralnervous systems. In the central nervous system (CNS), GDNF has beenshown to prevent the axotomy-induced death of mammalian facial andspinal cord motor neurons (Li et al., Proceedings Of The NationalAcademy Of Sciences, U.S.A., 92, 9771-9775, 1995; Oppenheim et al.,Nature, 373, 344-346, 1995; Yan et al., Nature, 373, 341-344, 1995;Henderson et al., Science, 266, 1062-1064, 1994; Zurn et al.,Neuroreport, 6, 113-118, 1994), and to rescue developing avian motorneurons from natural programmed cell death (Oppenheim et al., 1995supra). Local administration of GDNF has been shown to protect nigraldopaminergic neurons from axotomy-induced (Kearns and Gash, BrainResearch, 672, 104-111, 1995; Beck et al., Nature, 373, 339-341, 1995)or neurotoxin-induced degeneration (Sauer et al., Proceedings Of TheNational Academy Of Sciences U.S.A., 92, 8935-8939, 1995; Tomac et al.,Nature, 373, 335-339, 1995). In addition, local administration of GDNFhas been shown to induce sprouting from dopaminergic neurons, increaselevels of dopamine, noradrenaline, and serotonin, and improve motorbehavior (Tomac et al., 1995 supra).

[0005] More recently, GDNF has been reported to be a potential trophicfactor for brain noradrenergic neurons and Purkinje cells. Grafting offibroblasts ectopically expressing GDNF prevented6-hydroxydopamine-induced degeneration and promoted the phenotype ofadult noradrenergic neurons in vivo (Arenas et al., Neuron, 15,1465-1473, 1995), while exogeneously applied GDNF effectively promotedsurvival and morphological differentiation of embryonic Purkinje cellsin vitro (Mount et al., Proceedings Of The National Academy Of SciencesU.S.A., 92, 9092-9096, 1995). In the peripheral nervous system, GDNF hasbeen shown to promote the survival of neurons in nodose, ciliary, andsympathetic ganglia, as well as small populations of embryonic sensoryneurons in dorsal root ganglia (DRG) and trigeminal ganglia (Trupp etal., Journal Of Cell Biology, 130, 137-148, 1995; Ebendal et al.,Journal Of Neuroscience Research, 40, 276-284, 1995; Oppenheim et al.,1995 supra; Yan et al., 1995 supra; Henderson et al., 1994 supra). GDNFhas also been reported to enhance the expression of vasoactiveintestinal peptide and preprotachykinin-A mRNA in cultured superiorcervical ganglion (SCG) neurons, and thus effects the phenotype of SCGneurons and induces bundle-like sprouting (Trupp et al., 1995 supra).

[0006] Expression of GDNF has been observed in a number of differentcell types and structures of the nervous system. In the CNS, GDNF mRNAexpression has been observed by reverse transcriptase polymerase chainreaction (RT-PCR) in both developing and adult rat striatum, the majortarget of nigral dopaminergic innervation, and widely in other regions,including hippocampus, cortex, thalamus, septum, cerebellum, spinalcord, and medulla oblongata (Arenas et al., supra 1995; Poulsen et al.,Neuron, 13, 1245-1252, 1994; Springer et al., Experimental Neurology,127, 167-170, 1994; Stroemberg et al., Experimental Neurology, 124,401-412, 1993; Schaar et al., Experimental Neurology, 124, 368-371,1993). In human, GDNF transcripts have also been detected in striatum,with highest level in the caudate and lower levels in the putamen.Detectable levels are also found in hippocampus, cortex, and spinalcord, but not in cerebellum (Schaar et al., Experimental Neurology, 130,387-393, 1994; Springer et al., 1994 supra). In the periphery, GDNF mRNAexpression has been reported in DRG and SCG of postnatal day 1 rats,sciatic nerve, and primary cultures of neonatal Schwann cells (Trupp etal., 1995 supra; Hoffer et al., Neuroscience Letters, 182, 107-111,1994; Henderson et al., 1994 supra; Springer et al., 1994 supra). Inaddition, recent studies have shown that GDNF transcripts are alsowidely expressed in peripheral non-neuronal organs, including postnataltestis and kidney, embryonic whisker pad, stomach, and skin. Expressioncan be detected at lower levels in embryonic muscle, adrenal gland andlimb bud, and in postnatal lung, liver and ovary (Trupp et al., 1995supra; Henderson et al., 1994 supra). So far, however, the biologicalsignificance of the non-neuronal expression of GDNF is not clear.

[0007] Detailed descriptions of the preparation and characterization ofGDNF protein products may be found in U.S. patent application Ser. No.08/182,183 filed May 23, 1994 and its parent applications (also seePCT/US92/07888, WO 93/06116 filed Sep. 17, 1992 and European PatentApplication No. 92921022.7, Publication No. EP 610 254) the disclosuresof which are hereby incorporated by reference. Additional GDNF proteinproducts are described in pending U.S. patent application Ser. No.08/535,681 filed Sep. 28, 1995, the disclosure of which is herebyincorporated by reference. As used herein, the term “GDNF proteinproduct” includes biologically active synthetic or recombinant GDNFproteins and analogs, as well as chemically modified derivatives thereofGDNF analogs include deletion variants such as truncated GDNF proteins,as well as insertion and substitution variants of GDNF. Also includedare GDNF proteins that are substantially homologous to the human GDNFprotein.

[0008] GDNF Therapy

[0009] GDNF therapy is helpful in the treatment of nerve damage causedby conditions that compromise the survival and/or proper function of oneor more types of nerve cells. Such nerve damage may occur from a widevariety of different causes. Nerve damage may occur to one or more typesof nerve cells by: (1) physical injury, which causes the degeneration ofthe axonal processes and/or nerve cell bodies near the site of injury;(2) temporary or permanent cessation of blood flow to parts of thenervous system, as in stroke; (3) intentional or accidental exposure toneurotoxins, such as chemotherapeutic agents (e.g., cisplatinum) for thetreatment of cancer or dideoxycytidine (ddC) for the treatment of AIDS;(4) chronic metabolic diseases, such as diabetes or renal dysfunction;or (5) neurodegenerative diseases such as Parkinson's disease,Alzheimer's disease, and amyotrophic lateral sclerosis (ALS), whichresult from the degeneration of specific neuronal populations.

[0010] Several studies indicate that GDNF therapy is particularlyhelpful in the treatment of neurodegenerative conditions such as thedegeneration of the dopaminergic neurons of the substantia nigra inParkinson's disease. The only current treatments for Parkinson's diseaseare palliative, aiming at increasing dopamine levels in the striatum.The expected impact of GDNF therapy is not simply to produce an increasein the dopaminergic neurotransmission at the dopaminergic nerveterminals in the striatum (which will result in a relief of thesymptoms), but also to slow down, or even stop, the progression of thedegenerative processes and to repair the damaged nigrostriatal pathwayand restore its function. GDNF may also be used in treating other formsof damage to or improper function of dopaminergic nerve cells in humanpatients. Such damage or malfunction may occur in schizophrenia andother forms of psychosis. The only current treatments for suchconditions are symptomatic and require drugs which act upon dopaminereceptors or dopamine uptake sites, consistent with the view that theimproper functioning of the dopaminergic neurons which innervate thesereceptor-bearing neuronal populations may be involved in the diseaseprocess.

[0011] Receptors

[0012] A number of receptors which mediate binding and response toprotein factors have been characterized and molecularly cloned,including receptors for insulin, platelet derived growth factor,epidermal growth factor and its relatives, the fibroblast growthfactors, various interleukins, hematopoietic growth factors and ciliaryneurotrophic factor (U.S. Pat. No. 5,426,177). Study results indicatethat some receptors can bind to multiple (related) growth factors, whilein other cases the same factor can bind and activate multiple (related)receptors (e.g., Lupu et al., Science, 249:1552-1555, 1990; Dionne etal., EMBO J., 9:2685-2692, 1990; Miki et al., Science, 251:72-75, 1991).Most receptors can broadly be characterized as having an extracellularportion or domain responsible for specifically binding a protein factor,a transmembrane domain which spans the cell membrane, and anintracellular domain that is often involved in initiating signaltransduction upon binding of the protein factor to the receptor'sextracellular portion. Although many receptors are comprised of a singlepolypeptide chain, other receptors apparently require two or moreseparate subunits in order to bind to their protein factor withhigh-affinity and to allow functional response following binding (e.g.,Hempstead et al., Science, 243:373-375, 1989; Hibi et al., Cell,63:1149-1157, 1990).

[0013] The extracellular and intracellular portions of a given receptormay share common structural motifs with the corresponding regions ofother receptors, suggesting evolutionary and functional relationshipsbetween different receptors. These relationships can often be quitedistant and may simply reflect the repeated use of certain generaldomain structures. For example, a variety of different receptors thatbind unrelated factors make use of “immunoglobulin” domains in theirextracellular portions, while other receptors utilize “cytokinereceptor” domains in their factor-binding regions (e.g., Akira et al.,The FASEB J., 4:2860-2867, 1990). A large number of receptors withdistinct extracellular binding domains (which thus bind differentfactors) contain related intracytoplasmic domains encodingtyrosine-specific protein kinases that are activated in response tofactor binding (e.g., Ullrich and Schlessinger, Cell, 61:203-212, 1990).The mechanisms by which factor-binding “activates” the signaltransduction process is poorly understood, even in the case of receptortyrosine kinases. For other receptors, in which the intracellular domainencodes a domain of unknown function or in which the binding componentassociates with a second protein of unknown function (e.g., Hibi et al.,Cell, 63:1149-1157, 1990), activation of signal transduction is not wellcharacterized.

[0014] The mode of action of GDNF in vivo is not clearly elucidated inthe art, in part due to the absence of information on a receptor forGDNF. Two groups have independently found that striatum injected[¹²⁵I]-labeled GDNF can be retrogradely transported by dopaminergicneurons in the substantia nigra (Tomac et al., Proceedings Of TheNational Academy Of Sciences Of The United States Of America. 92,8274-8278, 1995; Yan et al., 1995 supra). Retrograde transport of[¹²⁵I]-GDNF by spinal cord motor neurons, DRG sensory neurons andneurons in the B layer of retina ganglia was also been observed. Theseretrograde transport phenomena can all be specifically inhibited by100-fold or higher concentrations of unlabeled GDNF, suggesting asaturable, receptor-mediated transport process. In vitro, recombinantGDNF has been shown to enhance the survival and promote dopamine uptakeof cultured dopaminergic neurons at very low concentrations. Theobserved half-maximal effective concentration (EC₅₀) of GDNF on theseneurons is 0.2 to 1.6 pM (Lin et al., 1993 supra). GDNF has also beenshown to support the survival of dissociated motor neurons at lowconcentrations. The reported EC₅₀ of GDNF on motor neurons, in a 5 to 10fM range, is even lower than that on dopaminergic neurons (Henderson etal., 1994 supra).

[0015] Taken together, these observations indicate that receptor(s) forGDNF expressed in these cells have very high ligand binding affinities.Similar to members of the TGF-B family, the widely diversified tissuedistribution and varied biological function of GDNF on differentpopulations of cells suggest that different types of receptor(s) forGDNF or receptor complexes may exist. Saturation steady-state andcompetitive binding of [¹²⁵I]-GDNF to E10 chick sympathetic neurons hasshown that these neurons express GDNF binding sites differing from thoseobserved in dopaminergic and motor neurons. The half maximal saturationconcentration and the half-maximal inhibition concentration of GDNF onthese binding sites is in the range of 1 to 5 nM (Trupp et al., 1995supra). Similarly, the EC₅₀ of GDNF in supporting the survival ofsympathetic neurons from PI rat SCG has also been reported to be in thenanomolar range (Trupp et al., 1995 supra).

[0016] To better understand the mechanism by which GDNF activates signaltransduction to exert its affects on cells, it would be beneficial toidentify the receptor(s) which mediate binding and response to thisprotein factor. It would also be beneficial for GDNF therapy to identifyand make possible the production of accessory molecules which providefor or enhance GDNF signal transduction. Moreover, the identification ofa protein receptor for GDNF would provide powerful applications indiagnostic uses, for example, as an aid in determining if individualswould benefit from GDNF protein therapy. Furthermore, the proteinreceptor for GDNF could be a key component in an assay for identifyingadditional molecules which bind to the receptor and result in desiredbiological activity.

SUMMARY OF THE INVENTION

[0017] The present invention provides nucleic acid sequences whichencode a neurotrophic factor receptor protein having an amino acidsequence as depicted in FIGS. 2 and 4 (SEQ. ID. NOs.: 2 and 4) as wellas biologically equivalent analogs. The neurotrophic factor receptorprotein and protein products of the present invention are designatedherein as glial cell line-derived neurotrophic factor receptor (GDNFR)protein and protein products. The novel GDNFRs are functionallycharacterized by the ability to bind GDNF specifically and with highaffinity, and to act as part of a molecular complex which mediates orenhances the signal transduction affects of GDNF. GDNFR protein productsare typically provided as a soluble receptor protein and in asubstantially purified form.

[0018] In one aspect, the present invention provides for the productionof GDNFR protein products by recombinant genetic engineering techniques.In an alternative embodiment, the GDNFR proteins are synthesized bychemical techniques, or a combination of the recombinant and chemicaltechniques.

[0019] In another aspect of the present invention, the GDNFR proteinsmay be made in glycosylated or non-glycosylated forms. Derivatives ofGDNFR protein typically involve attaching the GDNFR protein to a watersoluble polymer. For example, the GDNFR protein may be conjugated to oneor more polyethylene glycol molecules to decrease the precipitation ofthe GDNFR protein product in an aqueous environment.

[0020] Yet another aspect of the present invention includes the variouspolynucleotides encoding GDNFR proteins. These nucleic acid sequencesare used in the expression of GDNFR in a eukaryotic or prokaryotic hostcell, wherein the expression product or a derivative thereof ischaracterized by the ability to bind to GDNF and thereby form a complexcapable of mediating GDNF activity, such as increasing dopamine uptakeby dopaminergic cells. The polynucleotides may also be used in celltherapy or gene therapy applications. Suitable nucleic acid sequencesinclude those specifically depicted in the Figures as well as degeneratesequences, naturally occurring allelic variations and modified sequencesbased on the present invention.

[0021] Exemplary nucleic acid sequences include sequences encoding aneurotrophic factor receptor protein comprising an amino acid sequenceas depicted in FIGS. 2 and 4 (SEQ ID NOs. 2 and 4) capable of complexingwith glial cell line-derived neurotrophic factor (GDNF) and mediatingcell response to GDNF, and biologically equivalent analogs thereof. Suchsequences include: (a) a sequence set forth in FIG. 1 (SEQ ID NO. 1)comprising nucleotides encoding Met¹ through Ser⁴⁶⁵ or FIG. 3 (SEQ IDNO. 3) comprising nucleotides encoding Met¹ through Ser⁴⁶⁸ encoding aneurotrophic factor receptor (GDNFR) capable of complexing with glialcell line-derived neurotrophic factor (GDNF) and mediating cell responseto GDNF; (b) a nucleic acid sequence which (1) hybridizes to acomplementary sequence of (a) and (2) encodes an amino acid sequencewith GDNFR activity; and (c) a nucleic acid sequence which but for thedegeneracy of the genetic code would hybridize to a complementarysequence of (a) and (2) encodes an amino acid sequence with GDNFRactivity. Also disclosed herein are vectors such nucleic acid sequenceswherein the sequences typically are operatively linked to one or moreoperational elements capable of effecting the amplification orexpression of the nucleic acid sequence. Host cells containing suchvectors are also contemplated. Typically, the host cell is selected frommammalian cells and bacterial cells, such as a COS-7 cell or E. coli,respectively.

[0022] A further aspect of the present invention involves vectorscontaining the polynucleotides encoding GDNFR proteins operativelylinked to amplification and/or expression control sequences. Bothprokaryotic and eukaryotic host cells may be stably transformed ortransfected with such vectors to express GDNFR proteins. The presentinvention further includes the recombinant production of a GDNFR proteinwherein such transformed or transfected host cells are grown in asuitable nutrient medium, and the GDNFR expressed by the cells is,optionally, isolated from the host cells and/or the nutrient medium. Thepresent invention further includes the use of polynucleotides encodingGDNFR and vectors containing such polynucleotides in gene therapy orcell therapy.

[0023] The host cell may also be selected for its suitability to humanimplantation, wherein the implanted cell expresses and secretes aneurotrophic factor receptor of the present invention. The host cellalso may be enclosed in a semipermeable membrane suitable for humanimplantation. The host cell may be transformed or transfected ex vivo.An exemplary device for treating nerve damage involves: (a) asemipermeable membrane suitable for implantation; and (b) cellsencapsulated within the membrane, wherein the cells express and secretea neurotrophic factor receptor as disclosed herein. The membrane isselected from a material that is permeable to the neurotrophic factorreceptor protein but impermeable to materials detrimental to theencapsulated cells.

[0024] Methods for the recombinant production of a neurotrophic factorreceptor of the present invention are also disclosed. An exemplarymethods involves: (a) culturing a host cell containing a nucleic acidsequence encoding a neurotrophic factor receptor of the presentinvention, such as an amino acid sequence depicted in FIGS. 2 and 4 (SEQID NOs. 2 and 4) capable of complexing with glial cell line-derivedneurotrophic factor (GDNF) and mediating cell response to GDNF, orbiologically equivalent analogs thereof; (b) maintaining said host cellunder conditions suitable for the expression of said neurotrophic factorreceptor by said host cell; and (c) optionally, isolating saidneurotrophic factor receptor expressed by said host cell. The host cellmay be a prokaryotic cell or a eukaryotic cell. If bacterial expressionis involved, the method may further include the step of refolding theneurotrophic factor receptor.

[0025] The present invention includes an isolated and purified proteincomprising an amino acid sequence as depicted in FIGS. 2 and 4 (SEQ IDNOs. 2 and 4) capable of complexing with glial cell line-derivedneurotrophic factor (GDNF) and mediating cell response to GDNF, andbiologically equivalent analogs thereof. Exemplary analogs include, butare not limited to, proteins comprising the amino acid sequence Ser¹⁸through Pro⁴⁴⁶, Asp²⁵ through Leu⁴⁴⁷ and Cys²⁹ through Cys⁴⁴² asdepicted in FIG. 2 (SEQ ID NO:2) as well as proteins comprising theamino acid sequence Met¹⁷ through Pro⁴⁴⁹ and Cys²⁹ through Cys⁴⁴³ asdepicted in FIG. 4 (SEQ ID NO:4). The proteins of the present inventionmay be glycosylated or non-glycosylated and may be produced byrecombinant technology or chemical synthesis. The present inventionfurther includes nucleic acid sequences encoding a receptor proteincomprising such amino acid sequences.

[0026] Also disclosed herein are pharmaceutical compositions comprisinga protein receptor of the present invention in combination with apharmaceutically acceptable carrier. A variety of other formulationmaterials may be used to facilitate manufacture, storage, handling,delivery and/or efficacy.

[0027] Another aspect of the present invention includes the therapeuticuse of GDNFR genes and proteins. For example, a circulating or solubleGDNFR protein product may be used alone or in conjunction with GDNF intreating disease of or injury to the nervous system by enhancing theactivity of transmembrane signaling of GDNF. Thus, the proteins andpharmaceutical compositions of the present invention may be used intreating improperly functioning dopaminergic nerve cells, Parkinson'sdisease, Alzheimer's disease and amyotrophic lateral sclerosis.Alternatively, a recombinant GDNFR gene may be inserted in the cells oftissues which would benefit from increased sensitivity to GDNF, such asmotor neurons in patients suffering from amyotrophic lateral sclerosis.In yet another embodiment, GDNFR may be used to block GDNF activity incases where GDNF activity is thought to be detrimental. The GDNFR may beused to verify that observed effects of GDNF are due to the GDNFR.

[0028] In another aspect of the invention, GDNFR probes may be used toidentify cells and tissues which are responsive to GDNF in normal ordiseased states. Alternatively, the probes may be used to detect anaberrancy of GDNFR expression in a patient suffering from a GDNF-relateddisorder.

[0029] In a further aspect of the invention, GDNFR probes, includingnucleic acid as well as antibody probes, may be used to identifyGDNFR-related molecules. For example, the present invention provides forsuch molecules which form a complex with GDNFR and thereby participatein GDNFR function. As another example, the present invention providesfor receptor molecules which are homologous or cross-reactiveantigenically, but not identical to GDNFR.

[0030] The present invention also provides for the development of bothbinding and functional assays for GDNF based on the receptor. Forexample, assay systems for detecting GDNF activity may involve cellswhich express high levels of GDNFR, and which are therefore extremelysensitive to even very low concentrations of GDNF or GDNF-likemolecules. In yet another embodiment, soluble GDNFR may be used to bindor detect the presence of GDNF or GDNF-like molecules.

[0031] In addition, the present invention provides for experimentalmodel systems for studying the physiological role of GDNF. Such systemsinclude assays involving anti-GDNFR antibodies or oligonucleotide probesas well as animal models, such as transgenic animals which express highlevels of GDNFR and therefore are hypersensitive to GDNF or animalsderived using embryonic stem cell technology in which the endogenousGDNFR genes were deleted from the genome. An anti-GDNFR antibody willbinds a peptide portion of the neurotrophic factor receptor proteins.Antibodies include monoclonal and polyclonal antibodies. Alternatively,immunological tags for which antibodies already exist may be attached tothe GDNFR protein to aid in detection. Such tags include but are notlimited to Flag (IBI/Eastman Kodak) and myc sequences. Other tagsequences such as polyhistidine have also been used for detection andpurification on metal chelating columns.

[0032] Additional aspects and advantages of the invention will beapparent to those skilled in the art upon consideration of the followingdescription, which details the practice of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

[0033]FIG. 1 depicts a nucleic acid sequence (SEQ ID NO:1) encodinghuman glial cell line-derived neurotrophic factor receptor (GDNFR). Theamino acid sequence of the full length GDNFR protein is encoded bynucleic acids 540 to 1934.

[0034]FIG. 2 depicts the amino acid sequence (SEQ ID NO:2) of the fulllength human GDNFR protein.

[0035]FIG. 3 depicts a nucleic acid sequence (SEQ ID NO:3) encoding ratGDNFR. The amino acid sequence of the full length GDNFR protein isencoded by nucleic acids 302 to 1705.

[0036]FIG. 4 depicts the amino acid sequence (SEQ ID NO:4) of the fulllength rat GDNFR protein

[0037]FIG. 5 depicts the alignment and comparison of portions of GDNFRcDNA sequences produced in various clones as well as the consensussequence for human GDNFR.

[0038]FIG. 6 depicts the identification of Neuro-2A derived cell linesexpressing GDNFR.

[0039]FIGS. 7A and 7B depict the results of the equilibrium binding of[¹²⁵I]GDNF to cells expressing GDNFR.

[0040]FIG. 8 depicts the results of the chemical cross-linking of[¹²⁵I]GDNF to GDNFR and Ret Expressed in cells expressing GDNFR.

[0041]FIG. 9 depicts the results of the induction of c-Retautophosphorylation by GDNF in cells expressing GDNFR.

[0042]FIG. 10 depicts the results of the induction of c-Retautophosphorylation by GDNF and soluble GDNFR.

[0043]FIG. 11 depicts the results of the blocking of c-Retautophosphorylation by a Ret-Fc fusion protein.

[0044]FIG. 12 depicts the results of the induction of c-Retautophosphorylation by GDNF in motor neurons.

[0045]FIG. 13 depicts a model for GDNF signaling mediated by GDNFR andRet.

DETAILED DESCRIPTION OF THE INVENTION

[0046] Glial cell line-derived neurotrophic factor (GDNF) is a potentneurotrophic factor which exhibits a broad spectrum of biologicalactivities on a variety of cell types from both the central andperipheral nervous systems. It is a glycosylated, disulfide-linked dimerwhich is distantly related (less than 20% homology) to the transforminggrowth factor-β (TGF-β) superfamily. GDNF's ability to enhance thesurvival of dopaminergic neurons and other neuron populationsdemonstrates its therapeutic potential for the treatment of Parkinson'sdisease as well as other forms of nerve damage or malfunction.

[0047] In contrast to the extensive studies on the distribution andbioactivity of GDNF, there have been no reports on the identification ofa receptor or receptors which mediate binding of GDNF to a cell andthereby mediate intracellular signaling and a cell response. The presentinvention is based upon the discovery of a high affinity receptor firstfound on the surface of cultured retinal cells from postnatal rats.These receptors possess an estimated GDNF binding affinity comparable tothat of the receptors found in dopaminergic and motor neurons; midbraindopaminergic neurons (Lin et al., 1993 supra; Sauer et al., 1995 supra;Kearns and Gash, 1995 supra; Beck et al., 1995 supra; Tomac et al.,1995a supra), facial and spinal cord motor neurons (Li et al., 1995supra; Oppenheim et al., 1995 supra; Yan et al., 1995 supra; Zurn etal., 1994 supra; Henderson et al., 1994 supra). The receptor moleculehas been named GDNF receptor (GDNFR) since it is the first knowncomponent of a receptor system for GDNF. The present invention alsoprovides the first description of the expression cloning andcharacterization of GDNFR protein. Cells modified to express therecombinant receptor bind GDNF with high affinity.

[0048] Using a dopamine uptake assay and [¹²⁵I]-GDNF binding on culturedcells, high affinity receptors to GDNF were detected on the surface ofrat photoreceptor cells. As further described in the Examples, the studyof photoreceptor cells lead to the isolation of a cDNA clone byexpression cloning for GDNF receptor. The nucleic acid sequence forGDNFR encodes a protein of 468 amino acids with 31 cysteine residues andthree potential N-glycosylation sites. Next, a nucleic acid sequencefrom the rat cDNA clone was used to isolate its human homolog which wasfound to be nearly identical to the rat receptor at the amino acidlevel. The human GDNFR cDNA sequence encodes a protein of 465 aminoacids with the positions of all cysteine residues and potentialN-glycosylation sites conserved relative to the rat receptor. This highdegree of primary sequence conservation indicated an important role forthis receptor in the biological function of GDNF.

[0049] As discussed above, many receptors have three main domains: anextracellular or cell surface domain responsible for specificallybinding a protein factor; a transmembrane domain which spans the cell'smembrane; and an intracellular or cytoplasmic domain that is typicallyinvolved in initiating signal transduction when a protein factor bindsto the extracellular domain. It was determined, however, that GDNFR isunrelated in sequence or structural characteristics to any known protein(such as the consensus sequences found in either receptor kinases orcytokine receptors), lacks a cytoplasmic domain, lacks the C-terminalcharged residues characteristic of a transmembrane domain and isanchored to the cell membrane by glycosyl-phosphatidylinositol (GPI)linkage, as described in greater detail below. Although the absence ofan intracellular catalytic domain precluded a direct role intransmembrane signaling, the high binding affinity and strongevolutionary sequence conservation further suggested that this receptorwas important for GDNF function.

[0050] Because GDNFR lacks a cytoplasmic domain, it was thought thatthis receptor must act in conjunction with one or more accessorymolecules which play a role in transmembrane signaling. It was thendiscovered that transgenic mice which lack the gene for GDNF die andhave no kidneys. Transgenic mice which lack the gene for c-retproto-oncogene (Schuchardt, et al., Nature, 367, 380-383, 1994) werefound to have a similar phenotype. The c-ret proto-oncogene encodes areceptor tyrosine kinase (RTK) whose normal function had not yet beendetermined. All RTKs have a similar topology: they possess anextracellular ligand-binding domain, a transmembrane domain and acytoplasmic segment containing the catalytic protein-tyrosine kinasedomain. Binding of a ligand leads to the activation of the kinase domainand phosphorylation of specific substrates in the cell that mediateintracellular signaling. The present invention involves the discoverythat a soluble form of GDNFR may be used to mediate the binding of GDNFto the c-ret proto-oncogene and thereby elicit a cellular response toGDNF as well as modify its cell-type specificity.

[0051] Similar species, called “receptor alpha” components, provideligand binding specificity but do not have the capacity to transducesignal on their own. Such components are found in the ciliaryneurotrophic factor (CNTF) and interleukin-6 (IL-6) receptor systems.Like GDNFR, and in contrast to IL-6 receptor, CNTF receptor binds itsligand with high affinity, has a hydrophobic C-terminus, no cytoplasmicdomain, and is anchored to the cell membrane by GPI linkage (Davis etal., 1991). In order to mediate signal transduction, CNTF binds first toCNTF receptor, creating a complex which is able to bind gp130. Thisinactive complex then binds to LIF receptor to form the active signalingcomplex (Davis, et al., Science, 260, 1805-1807, 1993). As with thepresent invention, CNTF receptor (the ligand specific binding component)must be present for signaling to occur but it need not be membrane bound(Economides et al., Science, 270, 1351-1353, 1995).

[0052] As further described below, GDNFR protein may be anchored to acell surface, or it may be provided in a soluble form. In either case,GDNFR protein forms a ligand complex with GDNF, and the ligand complexbinds to cell surface receptor to effectuate intracellular signaling.Thus, a soluble form of GDNFR may be used to potentiate the action ofGDNF and/or modify its cell-type specificity.

[0053] GDNFR is unrelated to any known receptor. There are no apparentmatches in the GenBank and Washington University-Merck databases forrelated sequences. An expressed sequence tag (EST) found in theWashington University-Merck EST database shows 75% homology to a smallportion of the coding region of GDNFR (approximately 340 nucleotides ofthe 521 nucleotides of sequence generated from the 5′ end of the clone).This clone (GenBank accession #H12981) was isolated from an oligo-dTprimed human infant brain library and cloned directionally into theLafinid BA vector (Hillier, L. et al, unpublished data). The 3′ end ofthe #H12981 clone has been sequenced, but it exhibits no homology to anypart of the GDNFR. The appearance of homology between this #H12981 cloneand GDNFR over a short region, which homology then disappears, suggeststhat the #H12981 clone represents an unspliced transcript, or cloningartifact rather than a bona fide cDNA transcript.

[0054] Thus, the present invention enables the cloning of GDNFR proteinby providing a method for selecting target cells which express GDNFR. Byproviding a means of enriching for GDNFR encoding sequences, the presentinvention further provides for the purification of GDNFR protein and thedirect cloning of GDNFR-encoding DNA. The present description of theGDNFR nucleic acid and amino acid sequences provides the informationneeded to reproduce these entities as well as a variety of GDNFRanalogs. With this information, GDNFR protein products may be isolatedor generated by any means known to those skilled in the art. A varietyof means for the recombinant or synthetic production of GDNFR proteinare disclosed.

[0055] As used herein, the term “GDNFR protein product” includesbiologically active purified natural, synthetic or recombinant GDNFR,GDNFR analogs (i.e., GDNFR homologs and variants involving insertion,substitution and deletion variations), and chemically modifiedderivatives thereof. GDNFR analogs are substantially homologous to theGDNFR amino acid sequences set forth in FIGS. 2 and 4 (SEQ ID NOs:2 and4).

[0056] The term “biologically active”, as used herein, means that theGDNFR protein product demonstrates high affinity binding to GDNF andmediates or enhances GDNF-induced signal transduction. Using the presentdisclosure, it is well within the ability of those of ordinary skill inthe art to determine whether a GDNFR polypeptide analog hassubstantially the same biological activity as the GDNFR protein productsset forth in FIGS. 2 and 4.

[0057] The term “substantially homologous” amino acid sequence, as usedherein, refers to an amino acid sequence sharing a degree of“similarity” or homology to the GDNFR amino acid sequences set forth inFIGS. 2 and 4 such that the homologous sequence has a biologicalactivity or function similar to that described for these GDNFR aminoacid sequences. It will be appreciated by those skilled in the art, thata relatively large number of individual or grouped amino acid residuescan be changed, positionally exchanged (e.g.s, reverse ordered orreordered) or deleted entirely in an amino acid sequence withoutaffecting the three dimensional configuration or activity of themolecule. Such modifications are well within the ability of one skilledin the art following the present disclosure. The identification andmeans of providing such modified sequences are described in greaterdetail below. It is preferable that the degree of homology of asubstantially homologous protein (peptide) is equal to or in excess of70% (i.e., a range of from 70% to 100% homology). Thus, a preferable“substantially homologous” GDNFR amino acid sequence may have a degreeof homology greater than or equal to 70% of the amino acid sequences setforth in SEQ ID NOs:2 and 4. More preferably the degree of homology maybe equal to or in excess of 85%. Even more preferably it is equal to orin excess of 90%, or most preferably it is equal to or in excess of 95%.

[0058] The percentage of homology as described herein is calculated asthe percentage of amino acid residues found in one protein sequencewhich align with identical or similar amino acid residues in the secondprotein sequence. Thus, in the case of GDNFR homology, the degree ofsequence homology may be determined by optimally aligning the amino acidresidues of the comparison molecule to those of a reference GDNFRpolypeptide, such as depicted in SEQ ID NOs: 2 and 4 or those encoded bythe nucleic acid sequences depicted in the Figures, to maximize matchesof residues between the two sequences. It will be appreciated by thoseskilled in the art that such alignment may include appropriateconservative residue substitutions and will disregard truncations andinternal deletions or insertions of the comparison sequence byintroducing gaps as required; see, for example Dayhoff, Atlas of ProteinSequence and Structure Vol. 5, wherein an average of three or four gapsin a length of 100 amino acids may be introduced to assist in alignment(p. 124, National Biochemical Research Foundation, Washington, D.C.,1972; the disclosure of which is hereby incorporated by reference). Onceso aligned, the percentage is determined by the number of alignedresidues in the comparison polypeptide divided by the total number ofresidues in the comparison polypeptide. It is further contemplated thatthe GDNFR sequences of the present invention may be used to form aportion of a fusion protein or chimeric protein which has, at least inpart, GDNFR activity. The alignment and homology of such a protein wouldbe determined using that portion of the fusion protein or chimericprotein which is related to GDNFR activity.

[0059] The sources of such substantially homologous GDNFR proteinsinclude the GDNFR proteins of other mammals which are expected to have ahigh degree of homology to the human GDNFR protein. For example, thedegree of homology between the rat and human GDNFR proteins disclosedherein is about 93%. Substantially homologous GDNFR proteins may beisolated from such mammals by virtue of cross-reactivity with antibodiesto the GDNFR amino acid sequences of SEQ ID NOs: 2 and 4. Alternatively,they may be expressed by nucleic acid sequences which are isolatedthrough hybridization with the gene or with segments of the geneencoding the GDNFR of SEQ ID NOs 2 and 4 or which hybridize to acomplementary sequence of the nucleic acid sequences illustrated in SEQID NOs: 2 and 4. Suitable hybridization conditions are described infurther detail below.

[0060] The novel GDNFR protein products are typically isolated andpurified to form GDNFR protein products which are substantially free ofunwanted substances that would detract from the use of the presentpolypeptides for an intended purpose. For example, preferred GDNFRprotein products may be substantially free from the presence of otherhuman (e.g., non-GDNFR) proteinaceous materials or pathological agents.Preferably, the GDNFR protein products are about 80% free of otherproteins which may be present due to the production technique used inthe manufacture of the GDNFR protein product. More preferably, the GDNFRprotein products are about 90% free of other proteins, particularlypreferably, about 95% free of other proteins, and most preferablyabout >98% free of other proteins. In addition, the present inventionfurnishes the unique advantage of providing polynucleotide sequences forthe manufacture of homogeneous GDNFR proteins.

[0061] A variety of GDNFR variants are contemplated, including addition,deletion and substitution variants. For example, a series of deletionvariants may be made by removing one or more amino acid residues fromthe amino and/or carboxy termini of the GDNFR protein. Using rules forthe prediction of signal peptide cleavage as described by von Heijne(von Heijne, Nucleic Acids Research, 14, 4683-4690, 1986), the firstamino acid residue of the GDNFR protein which might be involved in GDNFbinding is Ser¹⁸, as depicted in the full length amino acid sequence ofhuman GDNFR in FIG. 2 (SEQ ID NO:2). Amino acid residues Met¹ throughSer¹⁸ are in the amino-terminal hydrophobic region that is likely to bepart of a signal peptide sequence, and therefore, not be included in themature form of the receptor protein. Similarly, the last amino acidresidue of the GDNFR protein which is likely to be necessary for GDNFbinding is Ser⁴⁴⁶. Amino acid residues Leu⁴⁴⁷ through Ser⁴⁶⁵ are in thecarboxy-terminal hydrophobic region that is involved in the GPI linkageof the protein to the cell surface. Thus, it is contemplated that any orall of the residues from Met¹ through Ser¹⁸ and/or Leu⁴⁴⁷ through Ser⁴⁶⁵(as depicted in FIG. 2 (SEQ ID NO:2) may be removed from the proteinwithout affecting GDNF binding to the GDNFR protein, thereby leaving a“core” sequence of Ala¹⁹ through Pro⁴⁴⁶. Using known analysistechniques, it is further contemplated that N-terminal truncations mayinclude the removal of one or more amino acid residues up to andincluding Gly²⁴. Thus, GDNFR truncation analogs also may include thedeletion of one or more amino acid residues from either or both terminisuch that an amino acid sequence of Asp²⁵ through pro⁴⁴⁶ or Leu⁴⁴⁷ formsthe basis for a core molecule. Additional GDNFR analogs are contemplatedas involving amino acid residues Ser¹⁸ through Pro⁴⁴⁹ as depicted in theGDNFR amino acid sequence of FIG. 4 (SEQ ID NO:4), i.e., deleting one ormore amino acid residues from either or both termini involving thehydrophobic regions depicted as amino acid residues Met¹ through Ser¹⁸and/or Pro⁴⁴⁹ through Ser⁴⁶⁸.

[0062] In addition, it is contemplated that one or more amino acidresidues may be removed from either or both of the amino and carboxytermini until the first and last cysteine residues in the full lengthsequence are reached. It is advantageous to retain the cysteine residuesfor the proper intramolecular binding of the GDNFR protein. As depictedin the full length amino acid sequence of human GDNFR in FIG. 2 (SEQ IDNO:2), any or all of amino acid residues from Met¹ to Asp²⁸ may beremoved from the amino terminal without removing the first cysteineresidue which appears as Cys²⁹. Similarly, any or all of amino acidresidues from Gly⁴⁴³ to Ser⁴⁶⁵ may be removed from the carboxy terminalwithout removing the last cysteine residue which appears as Cys⁴⁴².Other GDNFR analogs may be made using amino acid residues Cys²⁹ throughCys⁴⁴³ as depicted in the GDNFR amino acid sequence of FIG. 4 (SEQ IDNO:4), i.e., deleting all or part of the terminal regions depicted asamino acid residues Met¹ through Asp²⁸ and/or Ser⁴⁴⁴ through Ser⁴⁶⁸

[0063] It will be appreciated by those skilled in the art that, for thesame reasons, it is contemplated that these identified amino acidresidues may be replaced, rather than deleted, without affecting thefunction of the GDNFR protein. Alternatively, these identified aminoacid residues may be modified by intra-residue insertions or terminaladditions without affecting the function of the GDNFR protein. In yetanother embodiment, a combination of one or more deletions,substitutions or additions may be made.

[0064] The present GDNFR proteins or nucleic acids may be used formethods of treatment, or for methods of manufacturing medicaments fortreatment. Such treatment includes conditions characterized by excessiveproduction of GDNFR protein, wherein the present GDNFRs, particularly insoluble form, may be used to complex to and therefore inactivate suchexcessive GDNF protein. This treatment may be accomplished by preparingsoluble receptor (e.g., use of the GDNF binding domain) or bypreparation of a population of cells containing such GDNFR, andtransplanting such cells into the individual in need thereof. Thepresent GDNFR protein products may also be used for treatment of thosehaving defective GDNF receptors. For example, one may treat anindividual having defective GDNFRs by preparation and delivery of asoluble receptor, or by preparation of a population of cells containingsuch non-defective GDNFR and transplanting such cells into anindividual. Or, an individual may have an inadequate number of GDNFreceptors, and cells containing such receptors may be transplanted inorder to increase the number of GDNF receptors available to anindividual. Such compositions may be used in conjunction with thedelivery of GDNF. It is also contemplated GDNFR protein products may beused in the treatment of conditions responsive to the activation of thec-ret receptor tyrosine kinase.

[0065] In yet another aspect of the present invention, a furtheradvantage to the novel compositions is the use of GDNFR to stabilizeGDNF protein pharmaceutical compositions. In another aspect of thepresent invention, a GDNFR may be used to screen compounds forantagonist activity.

[0066] Other aspects and advantages of the present invention will beapparent to those skilled in the art. For example, additional usesinclude new assay systems, transgenic animals and antibody production.

[0067] Study Models

[0068] The present invention provides for assay systems in which GDNFactivity or activities similar to GDNF activity resulting from exposureto a peptide or non-peptide compound may be detected by measuring anelicited physiological response in a cell or cell line which expressesthe GDNFR molecules of the present invention. A physiological responsemay comprise any of the biological effects of GDNF, including but notlimited to, dopamine uptake, extension of neurites, increased cellsurvival or growth, as well as the transcriptional activation of certainnucleic acid sequences (e.g. promoter/enhancer elements as well asstructural genes), GDNF-related processing, translation, orphosphorylation, and the induction of secondary processes in response toprocesses directly or indirectly induced by GDNF, to name but a few.

[0069] For example, a model system may be created which may be used tostudy the effects of excess GDNF activity. In such a system, theresponse of a cell to GDNF may be increased by engineering an increasednumber of GDNFRs on the cells of the model system relative to cellswhich have not been so modified. A system may also be developed toselectively provide an increased number of GDNFRs on cells whichnormally express GDNFRs. In order to ensure expression of GDNFR, theGDNFR gene may be placed under the control of a suitable promotersequence. It may be desirable to put the GDNFR gene under the control ofa constitutive and/or tissue specific promoter (including but notlimited to the CNS neuron specific enolase, neurofilament, and tyrosinehydroxylase promoter), an inducible promoter (such as themetallothionein promoter), the UV activated promoter in the humanimmunodeficiency virus long terminal repeat (Valeri et al., 1988, Nature333:78-81), or the CMV promoter (as contained in pCMX, infra) or adevelopmentally regulated promoter.

[0070] By increasing the number of cellular GDNFRs, the response toendogenous GDNF may be increased. If the model system contains little orno GDNF, GDNF may be added to the system. It may also be desirable toadd additional GDNF to the model system in order to evaluate the effectsof excess GDNF activity. Over expressing GDNF (or secreted GDNF) may beone method for studying the effects of elevated levels of GDNF on cellsalready expressing GDNFR.

[0071] GDNFR Therapies

[0072] In another aspect, certain conditions may benefit from anincrease in GDNF responsiveness. It may, therefore, be beneficial toincrease the number or binding affinity of GDNFRs in patients sufferingfrom conditions responsive to GDNF therapy. This could be achievedthrough gene therapy, whereby selective expression of recombinant GDNFRin appropriate cells is achieved, for example, by using GDNFR genescontrolled by tissue specific or inducible promoters or by producinglocalized infection with replication defective viruses carrying arecombinant GDNFR gene.

[0073] It is envisioned that conditions which will benefit from GDNFR orcombined GDNF/GDNFR delivery include, but are not limited to, motorneuron disorders including amyotrophic lateral sclerosis, neurologicaldisorders associated with diabetes, Parkinson's disease, Alzheimer'sdisease, and Huntington's chorea. Additional indications for the use ofGDNFR or combined GDNF/GDNFR delivery are described above and furtherinclude the treatment of: glaucoma or other diseases and conditionsinvolving retinal ganglion cell degeneration; sensory neuropathy causedby injury to, insults to, or degeneration of, sensory neurons;pathological conditions, such as inherited retinal degenerations andage, disease or injury-related retinopathies, in which photoreceptordegeneration occurs and is responsible for vision loss; and injury ordegeneration of inner ear sensory cells, such as hair cells and auditoryneurons for preventing and/or treating hearing loss due to variety ofcauses.

[0074] Transgenic Animals

[0075] In yet another aspect, a recombinant GDNFR gene may be used toinactivate or “knock out” the endogenous gene (e.g., by homologousrecombination) and thereby create a GDNFR deficient cell, tissue, oranimal. For example, a recombinant GDNFR gene may be engineered tocontain an insertional mutation which inactivates GDNFR. Such aconstruct, under the control of a suitable promoter, may be introducedinto a cell, such as an embryonic stem cell, by any conventionaltechnique including transfection, transduction, injection, etc. Cellscontaining the construct may then be selected, for example by G418resistance. Cells which lack an intact GDNFR gene are then identified(e. g., by Southern blotting or Northern blotting or assay ofexpression). Cells lacking an intact GDNFR gene may then be fused toearly embryo cells to generate transgenic animals deficient in GDNFR. Acomparison of such an animal with an animal not expressing endogenousGDNF would reveal that either the two phenotypes match completely orthat they do not, implying the presence of additional GDNF-like factorsor receptors. Such an animal may be used to define specific neuronalpopulations, or other in vivo processes, normally dependent upon GDNF.Thus, these populations or processes may be expected to be effected ifthe animal did not express GDNFR, and therefore, could not respond toGDNF.

[0076] Diagnostic Applications

[0077] According to the present invention, GDNFR probes may be used toidentify cells and tissues which are responsive to GDNF in normal ordiseased states. The present invention provides for methods foridentifying cells which are responsive to GDNF by detecting GDNFRexpression in such cells. GDNFR expression may be evidenced bytranscription of GDNFR mRNA or production of GDNFR protein. GDNFRexpression may be detected using probes which identify GDNFR nucleicacid or protein or by detecting “tag” sequences artificially added tothe GDNFR protein.

[0078] One variety of probe which may be used to detect GDNFR expressionis a nucleic acid probe, which may be used to detect GDNFR-encoding RNAby any method known in the art, including, but not limited to, in situhybridization, Northern blot analysis, or PCR related techniques.Nucleic acid products of the invention may be labeled with detectablemarkers (such as radiolabels and non-isotopic labels such as biotin) andemployed in hybridization processes to locate the human GDNFR geneposition and/or the position of any related gene family in a chromosomalmap. They may also be used for identifying human GDNFR gene disorders atthe DNA level and used as gene markers for identifying neighboring genesand their disorders. Contemplated herein are kits containing suchlabeled materials.

[0079] Polypeptide products of the invention may be “labeled” byassociation with a detectable marker substance or label (e.g., aradioactive isotope, a fluorescent or chemiluminescent chemical, anenzyme or other label available to one skilled in the art) to providereagents useful in detection and quantification of GDNF in solid tissueand fluid samples such as blood or urine. Such products may also be usedin detecting cells and tissues which are responsive to GDNF in normal ordiseased states.

[0080] Another possible assay for detecting the presence of GDNF in atest sample or screening for the presence of a GDNF-like moleculeinvolves contacting the test sample with a GDNFR peptide immobilized ona solid phase, thereby producing GDNFR-bound GDNF. The GDNFR-bound GDNFmay optionally be contacted with a detection reagent, such as a labeledantibody specific for GDNF, thereby forming a detectable product. Suchassays may be developed in the form of assay devices for analyzing atest sample. In a basic form, such devices include a solid phasecontaining or coated with GDNFR. A method for analyzing a test samplefor the presence of GDNF may involve contacting the sample to an assayreagent comprising GDNFR protein, wherein said GDNFR protein reacts withGDNF present in the test sample and produces a detectable reactionproduct indicative of the presence of GDNF.

[0081] The assay reagents provided herein may also be embodied as partof a kit or article of manufacture. Contemplated is an article ofmanufacture comprising a packaging material and one or more preparationsof the presently provided nucleic acid or amino acid sequences. Suchpackaging material will comprise a label indicating that the preparationis useful for detecting GDNF, GDNFR or GDNFR defects in a biologicalsample. As such, the kit may optionally include materials to carry outsuch testing, such as reagents useful for performing protein analysis,DNA or RNA hybridization analysis, or PCR analysis on blood, urine, ortissue samples.

[0082] Anti-GDNFR Antibody

[0083] According to the present invention, GDNFR protein, or fragmentsor derivatives thereof, may be used as an immunogen to generateanti-GDNFR antibodies. To further improve the likelihood of producing ananti-GDNFR immune response, the amino acid sequence of GDNFR may beanalyzed in order to identify portions of the molecule which may beassociated with increased immunogenicity. For example, the amino acidsequence may be subjected to computer analysis to identify surfaceepitopes which present computer-generated plots of hydrophilicity,surface probability, flexibility, antigenic index, amphiphilic helix,amphiphilic sheet, and secondary structure of GDNFR. Alternatively, theamino acid sequences of GDNFR from different species could be compared,and relatively non-homologous regions identified; these non-homologousregions would be more likely to be immunogenic across various species.

[0084] Also comprehended are polypeptide fragments duplicating only apart of the continuous amino acid sequence or secondary conformationswithin GDNFR, which fragments may possess one activity of mammalianGDNFR (e.g., immunological activity) and not others (e.g., GDNF proteinbinding activity). Thus, the production of antibodies can include theproduction of anti-peptide antibodies. The following exemplary peptideswere synthesized using GDNFR sequences: TABLE 1 GDNFR Peptides SJP-6H₂N-QSCSTKYRTL-COOH human GDNFR, AA 40-49 (SEQ ID NO:25) SJP-7H₂N-CKRGMKKEKN-COOH human GDNFR, AA 89-98 (SEQ ID NO:26) SJP-8H₂N-LLEDSPYEPV-COOH human GDNFR, AA 115-124 (SEQ ID NO:27) SJP-9H₂N-CSYEERERPN-COOH rat GDNFR, AA 233-242 (SEQ ID NO:28) SJP-10H₂N-PAPPVQTTTATTTT-COOH rat GDNFR, AA 356-369 (SEQ ID NO:29)

[0085] Peptides SJP-6, 7, and 8 are identical in rat and human GDNFR.Peptides SJP-9 and 10 are derived from the rat sequence and are each oneamino acid different from human. Both polyclonal and monoclonalantibodies may be made by methods known in the art using these peptidesor other portions of GDNFR.

[0086] Monoclonal antibodies directed against GDNFR may be prepared byany known technique which provides for the production of antibodymolecules by continuous cell lines in culture. For example, thehybridoma technique originally developed by Kohler and Milstein toproduce monoclonal antibodies (Nature, 256:495-497, 1975), as well asthe trioma technique, the human B-cell hybridoma technique (Kozbor etal., Immunology Today 4:72, 1983), the EBV-hybridoma technique (Cole etal., in “Monoclonal Antibodies and Cancer Therapy,” Alan R. Liss, Inc.pp. 77-96, 1985), and the like, may be used.

[0087] Human monoclonal antibodies or chimeric human-mouse (or otherspecies) monoclonal antibodies also may be prepared for therapeutic useand may be made by any of numerous techniques known in the art (e.g.,Teng et al., Proc. Natl. Acad. Sci. U.S.A., 80:7308-7312, 1983; Kozboret al., Immunology Today, 4:72-79, 1983; Olsson et al., Meth. Enzymol.,92:3-16, 1982). Chimeric antibody molecules may be prepared containing amouse antigen-binding domain with human constant regions (Morrison etal., Proc. Natl. Acad. Sci. U.S.A., 81:6851, 1984; Takeda et al.,Nature, 314:452, 1985).

[0088] Various procedures known in the art also may be used for theproduction of polyclonal antibodies. For the production of antibody,various host animals including, but not limited to, rabbits, mice, rats,etc., can be immunized by injection with GDNFR protein, or a fragment orderivative thereof. Various adjuvants may be used to increase theimmunological response, depending on the host species selected. Usefuladjuvants include, but are not limited to, Freund's (complete andincomplete), mineral gels such as aluminum hydroxide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and humanadjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacteriumparvum.

[0089] A molecular clone of an antibody to a GDNFR epitope also may beprepared by known techniques. Recombinant DNA methodology (see e.g.,Maniatis et al., Molecular Cloning, A Laboratory Manual, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., 1982) may be used toconstruct nucleic acid sequences which encode a monoclonal antibodymolecule, or antigen binding region thereof.

[0090] Antibody molecules may be purified by known techniques, e.g.,immunoabsorption or immunoaffinity chromatography, chromatographicmethods such as high performance liquid chromatography, or a combinationthereof, etc. The present invention provides for antibody molecules aswell as fragments of such antibody molecules. Antibody fragments whichcontain the idiotype of the molecule can be generated by knowntechniques. For example, such fragments include but are not limited to:the F(ab′)2 fragment which can be produced by pepsin digestion of theantibody molecule; the Fab′ fragments which can be generated by reducingthe disulfide bridges of the F(ab′)2 fragment, and the Fab fragmentswhich can be generated by treating the antibody molecule with papain anda reducing agent.

[0091] Such selective binding molecules may themselves be alternativesto GDNFR protein, and may be formulated as a pharmaceutical composition.

[0092] Recombinant Expression of GDNFR Protein

[0093] The present invention provides various polynucleotides encodingGDNFR proteins. The expression product or a derivative thereof ischaracterized by the ability to bind to GDNF specifically and with highaffinity so that further interactions with signaling molecules canoccur, thereby providing or enhancing GDNF activity such as increasingdopamine uptake by dopaminergic cells. The polynucleotides may also beused in cell therapy or gene therapy applications.

[0094] According to the present invention, novel GDNFR protein and DNAsequences coding for all or part of such receptors are provided. Novelnucleic acid sequences of the invention include sequences useful insecuring expression in procaryotic or eucaryotic host cells ofpolypeptide products having at least a part of the primary structuralconformation and one or more of the biological properties of recombinanthuman GDNFR. The nucleic acids may be purified and isolated, so that thedesired coding region is useful to produce the present polypeptides.Alternatively, the nucleic acid sequence may be used for diagnosticpurposes, as described more fully below. Exemplary DNA sequences of thepresent invention comprise nucleic acid sequences encoding the GDNFRamino acid sequences depicted in FIGS. 2 and 4 and set forth in SEQ. IDNOs:2 and 4. In addition, DNA sequences disclosed by the presentinvention specifically comprise: (a) any of the DNA sequences depictedin FIGS. 1 and 3 (and complementary strands); (b) a DNA sequence whichhybridizes (under hybridization conditions disclosed in the cDNA libraryscreening section below, or equivalent conditions or more stringentconditions) to the DNA sequence in subpart (a) or to fragments thereof;and (c) a DNA sequence which, but for the degeneracy of the geneticcode, would hybridize to the DNA sequence in subpart (a). Specificallycomprehended in parts (b) and (c) are genomic DNA sequences encodingallelic variant forms of human GDNFR and/or encoding GDNFR from othermammalian species, and manufactured DNA sequences encoding GDNFR,fragments of GDNFR, and analogs of GDNFR which DNA sequences mayincorporate codons facilitating transcription and translation ofmessenger RNA in microbial hosts. Such manufactured sequences mayreadily be constructed according to the methods known in the art as wellas the methods described herein.

[0095] Recombinant expression techniques, conducted in accordance withthe descriptions set forth herein or other known methods, may be used toproduce these polynucleotides and express the various GDNFR proteins.For example, by inserting a nucleic acid sequence which encodes a GDNFRprotein into an appropriate vector, one skilled in the art can readilyproduce large quantities of the desired nucleotide sequence. Thesequences can then be used to generate detection probes or amplificationprimers. Alternatively, a polynucleotide encoding a GDNFR protein can beinserted into an expression vector. By introducing the expression vectorinto an appropriate host, the desired GDNFR protein may be produced inlarge amounts.

[0096] As further described herein, there are numerous host/vectorsystems available for the propagation of nucleic acid sequences and/orthe production of GDNFR proteins. These include, but are not limited to,plasmid, viral and insertional vectors, and prokaryotic and eukaryotichosts. One skilled in the art can adapt a host/vector system which iscapable of propagating or expressing heterologous DNA to produce orexpress the sequences of the present invention.

[0097] By means of such recombinant techniques, the GDNFR proteins ofthe present invention are readily produced in commercial quantities withgreater purity. Furthermore, it will be appreciated by those skilled inthe art that, in view of the present disclosure, the novel nucleic acidsequences include degenerate nucleic acid sequences encoding the GDNFRproteins specifically set forth in the Figures, sequences encodingvariants of GDNFR proteins, and those nucleic acid sequences whichhybridize, preferably under stringent hybridization conditions, tocomplements of these nucleic acid sequences (see, Maniatis et. al.,Molecular Cloning (A Laboratory Manual); Cold Spring Harbor Laboratory,pages 387 to 389, 1982.) Exemplary stringent hybridization conditionsare hybridization in 4× SSC at 62-67° C., followed by washing in 0.1×SSC at 62-67° C. for approximately an hour. Alternatively, exemplarystringent hybridization conditions are hybridization in 45-55%formamide, 4× SSC at 40-45° C. DNA sequences which hybridize to thecomplementary sequences for GDNFR protein under relaxed hybridizationconditions and which encode a GDNFR protein of the present invention arealso included herein. Examples of such relaxed stringency hybridizationconditions are 4× SSC at 45-55° C. or hybridization with 30-40%formamide at 40-45° C.

[0098] Preparation of Polynucleotides Encoding GDNFR

[0099] Based upon the disclosure of the present invention, a nucleicacid sequence encoding a full length GDNFR polypeptide or a fragmentthereof may readily be prepared or obtained in a variety of ways,including, without limitation, chemical synthesis, cDNA or genomiclibrary screening, expression library screening, and/or PCRamplification of cDNA. These methods and others useful for preparingnucleic acid sequences are known in the art and are set forth, forexample, by Sambrook et al. (Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), byAusubel et al., eds (Current Protocols in Molecular Biology, CurrentProtocols Press, 1994), and by Berger and Kimmel (Methods in Enzymology:Guide to Molecular Cloning Techniques, vol. 152, Academic Press, Inc.,San Diego, Calif., 1987). Preferred nucleic acid sequences encodingGDNFR are mammalian sequences.

[0100] Chemical synthesis of a nucleic acid sequence which encodes aGDNFR protein can also be accomplished using methods known in the art,such as those set forth by Engels et al. (Angew. Chem. Intl. Ed.,28:716-734, 1989). These methods include, inter alia, thephosphotriester, phosphoramidite and H-phosphonate methods of nucleicacid sequence synthesis. A preferred method for such chemical synthesisis polymer-supported synthesis using standard phosphoramidite chemistry.Typically, the DNA encoding the desired polypeptide will be severalhundred base pairs (bp) or nucleotides in length. Nucleic acid sequenceslarger than about 100 nucleotides can be synthesized as severalfragments using these methods. The fragments can then be ligatedtogether to form a sequence for the expression of a full length GDNFRpolypeptide or a portion thereof.

[0101] Alternatively, a suitable nucleic acid sequence may be obtainedby screening an appropriate cDNA library (i.e., a library prepared fromone or more tissue source(s) believed to express the protein) or agenomic library (a library prepared from total genomic DNA). The sourceof the cDNA library is typically a tissue that is believed to expressGDNFR in reasonable quantities. Typically, the source of the genomiclibrary is any tissue or tissues from a mammalian species believed toharbor a gene encoding GDNFR. The library can be screened for thepresence of the GDNFR cDNA/gene using one or more nucleic acid probes(such as oligonucleotides, cDNA or genomic DNA fragments based upon thepresently disclosed sequences) that will hybridize selectively withGDNFR cDNA(s) or gene(s) present in the library. The probes typicallyused for such library screening usually encode a small region of theGDNFR nucleic acid sequence from the same or a similar species as thespecies from which the library was prepared. Alternatively, the probesmay be degenerate, as discussed herein.

[0102] Library screening is typically accomplished by annealing theoligonucleotide probe or cDNA to the clones in the library underconditions of stringency that prevent non-specific binding but permitbinding (hybridization) of those clones that have a significant level ofhomology with the probe or primer. Typical hybridization and washingstringency conditions depend in part on the size (i.e., number ofnucleotides in length) of the cDNA or oligonucleotide probe, and whetherthe probe is degenerate. The probability of obtaining a clone(s) is alsoconsidered in designing the hybridization solution (e.g., whether a cDNAor genomic library is being screened; if it is a cDNA library, theprobability that the cDNA of interest is present at a high level).

[0103] Where DNA fragments (such as cDNAs) are used as probes, typicalhybridization conditions include those as set forth in Ausubel et al.,eds., supra. After hybridization, the blot containing the library iswashed at a suitable stringency, depending on several factors such asprobe size, expected homology of probe to clone, type of library beingscreened, number of clones being screened, and the like. Examples ofstringent washing solutions (which are usually low in ionic strength andare used at relatively high temperatures) are as follows. One suchstringent wash is 0.015 M NaCl, 0.005 M NaCitrate and 0.1% SDS at 55-65°C. Another such stringent buffer is 1 mM Na₂EDTA, 40 mM NaHPO₄, pH 7.2,and 1% SDS at about 40-50° C. One other stringent wash is 0.2× SSC and0.1% SDS at about 50-65° C.

[0104] There are also exemplary protocols for stringent washingconditions where oligonucleotide probes are used to screen cDNA orgenomic libraries. For example, a first protocol uses 6× SSC with 0.05percent sodium pyrophosphate at a temperature of between about 35 and62° C., depending on the length of the probe. For example, 14 baseprobes are washed at 35-40° C., 17 base probes at 45-50° C., 20 baseprobes at 52-57° C., and 23 base probes at 57-63° C. The temperature canbe increased 2-3° C. where the background non-specific binding appearshigh. A second protocol uses tetramethylammonium chloride (TMAC) forwashing. One such stringent washing solution is 3 M TMAC, 50 mMTris-HCl, pH 8.0, and 0.2% SDS.

[0105] Another suitable method for obtaining a nucleic acid sequenceencoding a GDNFR protein is by polymerase chain reaction (PCR). In thismethod, poly(A)+RNA or total RNA is extracted from a tissue thatexpresses GDNFR. A cDNA is then prepared from the RNA using the enzymereverse transcriptase (i.e., RT-PCR). Two primers, typicallycomplementary to two separate regions of the GDNFR cDNA(oligonucleotides), are then added to the cDNA along with a polymerasesuch as Taq polymerase, and the polymerase amplifies the cDNA regionbetween the two primers.

[0106] Where the method of choice for preparing the nucleic acidsequence encoding the desired GDNFR protein requires the use ofoligonucleotide primers or probes (e.g., PCR, cDNA or genomic libraryscreening), the oligonucleotide sequences selected as probes or primersshould be of adequate length and sufficiently unambiguous so as tominimize the amount of non-specific binding that will occur duringlibrary screening or PCR amplification. The actual sequence of theprobes or primers is usually based on conserved or highly homologoussequences or regions from the same or a similar gene from anotherorganism, such as the rat nucleic acid sequence involved in the presentinvention. Optionally, the probes or primers can be fully or partiallydegenerate, i.e., contain a mixture of probes/primers, all encoding thesame amino acid sequence, but using different codons to do so. Analternative to preparing degenerate probes is to place an inosine insome or all of those codon positions that vary by species. Theoligonucleotide probes or primers may be prepared by chemical synthesismethods for DNA as described above.

[0107] GDNFR proteins based on these nucleic acid sequences encodingGDNFR, as well as mutant or variant sequences thereof, are alsocontemplated as within the scope of the present invention. Mutant orvariant sequences include those sequences containing one or morenucleotide substitutions, deletions, and/or insertions as compared tothe wild type sequence and that results in the expression of amino acidsequence variations as compared to the wild type amino acid sequence. Insome cases, naturally occurring GDNFR amino acid mutants or variants mayexist, due to the existence of natural allelic variation. GDNFR proteinsbased on such naturally occurring mutants or variants are also withinthe scope of the present invention. Preparation of synthetic mutantsequences is also well known in the art, and is described for example inWells et al. (Gene, 34:315, 1985) and in Sambrook et al., supra.

[0108] In some cases, it may be desirable to prepare nucleic acid and/oramino acid variants of naturally occurring GDNFR. Nucleic acid variants(wherein one or more nucleotides are designed to differ from thewild-type or naturally occurring GDNFR) may be produced using sitedirected mutagenesis or PCR amplification where the primer(s) have thedesired point mutations (see Sambrook et al., supra, and Ausubel et al.,supra, for descriptions of mutagenesis techniques). Chemical synthesisusing methods described by Engels et al., supra, may also be used toprepare such variants. Other methods known to the skilled artisan may beused as well. Preferred nucleic acid variants are those containingnucleotide substitutions accounting for codon preference in the hostcell that is to be used to recombinantly produce GDNFR. Other preferredvariants are those encoding conservative amino acid changes (e.g.,wherein the charge or polarity of the naturally occurring amino acidside chain is not altered substantially by substitution with a differentamino acid) as compared to wild type, and/or those designed to eithergenerate a novel glycosylation and/or phosphorylation site(s) on GDNFR,or those designed to delete an existing glycosylation and/orphosphorylation site(s) on GDNFR.

[0109] Vectors

[0110] The cDNA or genomic DNA encoding the desired GDNFR protein isinserted into a vector for further cloning (amplification of the DNA) orfor expression. Suitable vectors are commercially available, or thevector may be specially constructed. Possible vectors include, but arenot limited to, cosmids, plasmids or modified viruses, but the vectorsystem must be compatible with the selected host cell. Such vectorsinclude, but are not limited to, bacteriophages such as lambdaderivatives, or plasmids such as pBR322, pUC, or Bluescript® plasmidderivatives (Stratagene, La Jolla Calif.). The recombinant molecules canbe introduced into host cells via transformation, transfection,infection, electroporation, or other known techniques.

[0111] For example, the GDNFR-encoding nucleic acid sequence is insertedinto a cloning vector which is used to transform, transfect, or infectappropriate host cells so that many copies of the nucleic acid sequenceare generated. This can be accomplished by ligating a DNA fragment intoa cloning vector which has complementary cohesive termini. If thecomplementary restriction sites used to fragment the DNA are not presentin the cloning vector, the ends of the DNA molecules may beenzymatically modified. It also may prove advantageous to incorporaterestriction endonuclease cleavage sites into the oligonucleotide primersused in polymerase chain reaction to facilitate insertion of theresulting nucleic acid sequence into vectors. Alternatively, any sitedesired may be produced by ligating nucleotide sequences (linkers) ontothe DNA termini; these ligated linkers may comprise specific chemicallysynthesized oligonucleotides encoding restriction endonucleaserecognition sequences. In an alternative method, the cleaved vector andGDNFR-encoding nucleic acid sequence may be modified by homopolymerictailing. In specific embodiments, transformation of host cells withrecombinant DNA molecules that incorporate an isolated GDNFR gene, cDNA,or synthesized DNA sequence enables generation of multiple copies of thegene. Thus, the GDNFR-encoding nucleic acid sequence may be obtained inlarge quantities by growing transformants, isolating the recombinant DNAmolecules from the transformants and, when necessary, retrieving theinserted gene from the isolated recombinant DNA.

[0112] The selection or construction of the appropriate vector willdepend on 1) whether it is to be used for DNA amplification or for DNAexpression, 2) the size of the DNA to be inserted into the vector, and3) the host cell (e.g., mammalian, insect, yeast, fungal, plant orbacterial cells) to be transformed with the vector. Each vector containsvarious components depending on its function (amplification of DNA orexpression of DNA) and its compatibility with the intended host cell.For DNA expression, the vector components may include, but are notlimited to, one or more of the following: a signal sequence, an originof replication, one or more selection or marker genes, enhancerelements, promoters, a transcription termination sequence, and the like.These components may be obtained from natural sources or synthesized byknown procedures. The vectors of the present invention involve a nucleicacid sequence which encodes the GDNFR protein of interest operativelylinked to one or more amplification, expression control, regulatory orsimilar operational elements capable of directing, controlling orotherwise effecting the amplification or expression of theGDNFR-encoding nucleic acid sequence in the selected host cell.

[0113] Expression vectors containing GDNFR nucleic acid sequence insertscan be identified by three general approaches: (a) DNA-DNAhybridization; (b) the presence or absence of “marker” gene functions,and (c) the expression of inserted sequences. In the first approach, thepresence of a foreign nucleic acid sequence inserted in an expressionvector can be detected by DNA-DNA hybridization using probes comprisingsequences that are homologous to an inserted GDNFR-encoding nucleic acidsequence. In the second approach, the recombinant vector/host system canbe identified and selected based upon the presence or absence of certain“marker” gene functions (e.g., thymidine kinase activity, resistance toantibiotics, transformation phenotype, occlusion body formation inbaculovirus, etc.) caused by the insertion of a foreign nucleic acidsequence into the vector. For example, if a GDNFR-encoding nucleic acidsequence is inserted within the marker gene sequence of the vector,recombinants containing the GDNFR insert can be identified by theabsence of the marker gene function. In the third approach, recombinantexpression vectors can be identified by detecting the foreign proteinproduct expressed by the recombinant nucleic acid sequence. Such assayscan be based on the physical or functional properties of the expressedGDNFR protein product, for example, by binding of the GDNFR protein toGDNF or to an antibody which directly recognizes GDNFR.

[0114] Signal Sequence

[0115] The signal sequence may be a component of the vector, or it maybe a part of GDNFR DNA that is inserted into the vector. The nativeGDNFR DNA encodes a signal sequence at the amino terminus of the proteinthat is cleaved during post-translational processing of the protein toform the mature GDNFR protein. Included within the scope of thisinvention are GDNFR polynucleotides with the native signal sequence aswell as GDNFR polynucleotides wherein the native signal sequence isdeleted and replaced with a heterologous signal sequence. Theheterologous signal sequence selected should be one that is recognizedand processed, i.e., cleaved by a signal peptidase, by the host cell.For prokaryotic host cells that do not recognize and process the nativeGDNFR signal sequence, the signal sequence is substituted by aprokaryotic signal sequence selected, for example, from the group of thealkaline phosphatase, penicillinase, or heat-stable enterotoxin IIleaders. For yeast secretion, the native GDNFR signal sequence may besubstituted by the yeast invertase, alpha factor, or acid phosphataseleaders. In mammalian cell expression the native signal sequence issatisfactory, although other mammalian signal sequences may be suitable.

[0116] Origin of Replication

[0117] Expression and cloning vectors generally include a nucleic acidsequence that enables the vector to replicate in one or more selectedhost cells. In cloning vectors, this sequence is typically one thatenables the vector to replicate independently of the host chromosomalDNA, and includes origins of replication or autonomously replicatingsequences. Such sequences are well known for a variety of bacteria,yeasts, and viruses. The origin of replication from the plasmid pBR322is suitable for most Gram-negative bacteria and various origins (e.g.,SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors inmammalian cells. Generally, the origin of replication component is notneeded for mammalian expression vectors (for example, the SV40 origin isoften used only because it contains the early promoter).

[0118] Selection Gene

[0119] The expression and cloning vectors may contain a selection gene.This gene encodes a “marker” protein necessary for the survival orgrowth of the transformed host cells when grown in a selective culturemedium. Host cells that were not transformed with the vector will notcontain the selection gene, and therefore, they will not survive in theculture medium. Typical selection genes encode proteins that (a) conferresistance to antibiotics or other toxins, e.g., ampicillin, neomycin,methotrexate, or tetracycline; (b) complement auxotrophic deficiencies;or (c) supply critical nutrients not available from the culture medium.

[0120] Other selection genes may be used to amplify the gene which willbe expressed. Amplification is the process wherein genes which are ingreater demand for the production of a protein critical for growth arereiterated in tandem within the chromosomes of successive generations ofrecombinant cells. Examples of suitable selectable markers for mammaliancells include dihydrofolate reductase (DHFR) and thymidine kinase. Themammalian cell transformants are placed under selection pressure whichonly the transformants are uniquely adapted to survive by virtue of themarker present in the vector. Selection pressure is imposed by culturingthe transformed cells under conditions in which the concentration ofselection agent in the medium is successively changed, thereby leadingto amplification of both the selection gene and the DNA that encodesGDNFR. As a result, increased quantities of GDNFR are synthesized fromthe amplified DNA.

[0121] For example, cells transformed with the DHFR selection gene arefirst identified by culturing all of the transformants in a culturemedium that contains methotrexate, a competitive antagonist of DHFR. Anappropriate host cell when wild-type DHFR is used is the Chinese hamsterovary cell line deficient in DHFR activity (see, for example, Urlaub andChasin, Proc. Natl. Acad. Sci., U.S.A., 77(7): 4216-4220, 1980). Thetransformed cells are then exposed to increased levels of methotrexate.This leads to the synthesis of multiple copies of the DHFR gene, and,concomitantly, multiple copies of other DNA present in the expressionvector, such as the DNA encoding a GDNFR protein.

[0122] Promoter

[0123] The expression and cloning vectors of the present invention willtypically contain a promoter that is recognized by the host organism andoperably linked to the nucleic acid sequence encoding the GDNFR protein.Promoters are untranslated sequences located upstream (5′) to the startcodon of a structural gene (generally within about 100 to 1000 bp) thatcontrol the transcription and translation of a particular nucleic acidsequence, such as that encoding GDNFR. Promoters are conventionallygrouped into one of two classes, inducible promoters and constitutivepromoters. Inducible promoters initiate increased levels oftranscription from DNA under their control in response to some change inculture conditions, such as the presence or absence of a nutrient or achange in temperature. A large number of promoters, recognized by avariety of potential host cells, are well known. These promoters areoperably linked to the DNA encoding GDNFR by removing the promoter fromthe source DNA by restriction enzyme digestion and inserting the desiredpromoter sequence into the vector. The native GDNFR promoter sequencemay be used to direct amplification and/or expression of GDNFR DNA. Aheterologous promoter is preferred, however, if it permits greatertranscription and higher yields of the expressed protein as compared tothe native promoter, and if it is compatible with the host cell systemthat has been selected for use.

[0124] Promoters suitable for use with prokaryotic hosts include thebeta-lactamase and lactose promoter systems; alkaline phosphatase, atryptophan (trp) promoter system; and hybrid promoters such as the tacpromoter. Other known bacterial promoters are also suitable. Theirnucleotide sequences have been published, thereby enabling one skilledin the art to ligate them to the desired DNA sequence(s), using linkersor adaptors as needed to supply any required restriction sites.

[0125] Suitable promoting sequences for use with yeast hosts are alsowell known in the art. Yeast enhancers are advantageously used withyeast promoters. Suitable promoters for use with mammalian host cellsare well known and include those obtained from the genomes of virusessuch as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2),bovine papilloma virus, avian sarcoma virus, cytomegalovirus, aretrovirus, hepatitis-B virus and most preferably Simian Virus 40(SV40). Other suitable mammalian promoters include heterologousmammalian promoters, e.g., heat-shock promoters and the actin promoter.A promoter for possible use in the production of GDNFR proteins in CHOcells is SRa (see Takebe et al., Mol. Cell. Biol., 8(1): 466-472, 1988).A suitable expression vector is pDSRa2. The pDSRa2 plasmid constructscontaining the appropriate GDNFR cDNA may be prepared substantially inaccordance with the process described in the co-owned and copending U.S.patent application Ser. No. 501,904 filed Mar. 29, 1990 (also see,European Patent Application No. 90305433, Publication No. EP 398 753,filed May 18, 1990 and WO 90/14363 (1990), the disclosures of which arehereby incorporated by reference.

[0126] Additional promoters which may be of interest in controllingGDNFR expression include, but are not limited to: the SV40 earlypromoter region (Bemoist and Chambon, Nature, 290:304-310, 1981); theCMV promoter; the promoter contained in the 3′ long terminal repeat ofRous sarcoma virus (Yamamoto, et al., Cell, 22:787-797, 1980); theherpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci.U.S.A., 78:144-1445, 1981); the regulatory sequences of themetallothionine gene (Brinster et al., Nature, 296:39-42, 1982);prokaryotic expression vectors such as the beta-lactamase promoter(Villa-Kamaroff, et al., Proc. Natl. Acad. Sci. U.S.A., 75:3727-3731,1978); or the tac promoter (DeBoer, et al., Proc. Natl. Acad. Sci.U.S.A., 80:21-25, 1983). Also of interest are the following animaltranscriptional control regions, which exhibit tissue specificity andhave been utilized in transgenic animals: the elastase I gene controlregion which is active in pancreatic acinar cells (Swift et al., Cell,38:639-646, 1984; Omitz et al., Cold Spring Harbor Symp. Quant. Biol.50:399-409, 1986; MacDonald, Hepatology, 7:425-515, 1987); the insulingene control region which is active in pancreatic beta cells (Hanahan,Nature, 315:115-122, 1985); the immunoglobulin gene control region whichis active in lymphoid cells (Grosschedl et al., Cell, 38:647-658, 1984;Adames et al., Nature, 318:533-538, 1985; Alexander et al., Mol. Cell.Biol., 7:1436-1444, 1987); the mouse mammary tumor virus control regionwhich is active in testicular, breast, lymphoid and mast cells (Leder etal., Cell, 45:485-495, 1986), albumin gene control region which isactive in liver (Pinkert et al., Genes and Devel., 1:268-276, 1987); thealpha-fetoprotein gene control region which is active in liver (Krumlaufet al., Mol. Cell. Biol., 5:1639-1648, 1985; Hammer et al., Science,235:53-58, 1987); the alpha 1-antitrypsin gene control region which isactive in the liver (Kelsey et al., Genes and Devel., 1:161-171, 1987);the beta-globin gene control region which is active in myeloid cells(Mogram et al., Nature, 315:338-340, 1985; Kollias et al., Cell,46:89-94, 1986); the myclin basic protein gene control region which isactive in oligodendrocyte cells in the brain (Readhead et al., Cell,48:703-712, 1987); the myosin light chain-2 gene control region which isactive in skeletal muscle (Sani, Nature, 314:283-286, 1985); and thegonadotropic releasing hormone gene control region which is active inthe hypothalamus (Mason et al., Science, 234:1372-1378, 1986).

[0127] Enhancer Element

[0128] An enhancer sequence may be inserted into the vector to increasethe transcription of a DNA sequence encoding a GDNFR protein of thepresent invention by higher eukaryotes. Enhancers are cis-actingelements of DNA, usually about 10-300 bp in length, that act on thepromoter to increase its transcription. Enhancers are relativelyorientation and position independent. They have been found 5′ and 3′ tothe transcription unit. Several enhancer sequences available frommammalian genes are known (e.g., globin, elastase, albumin,alpha-feto-protein and insulin). Typically, however, an enhancer from avirus will be used. The SV40 enhancer, the cytomegalovirus earlypromoter enhancer, the polyoma enhancer, and adenovirus enhancers areexemplary enhancing elements for the activation of eukaryotic promoters.While an enhancer may be spliced into the vector at a position 5′ or 3′to GDNFR DNA, it is typically located at a site 5′ from the promoter.

[0129] Transcription Termination

[0130] Expression vectors used in eukaryotic host cells (yeast, fungi,insect, plant, animal, human, or nucleated cells from othermulticellular organisms) will also contain sequences necessary forterminating transcription and stabilizing the mRNA. Such sequences arecommonly available from the 5′ and occasionally 3′ untranslated regionsof eukaryotic DNAs or cDNAs. These regions contain nucleotide segmentstranscribed as polyadenylated fragments in the untranslated portion ofthe mRNA encoding GDNFR.

[0131] The construction of suitable vectors containing one or more ofthe above-listed components together with the desired GDNFR-encodingsequence is accomplished by standard ligation techniques. Isolatedplasmids or DNA fragments are cleaved, tailored, and religated in thedesired order to generate the plasmids required. To confirm that thecorrect sequences have been constructed, the ligation mixtures may beused to transform E. coli, and successful transformants may be selectedby known techniques, such as ampicillin or tetracycline resistance asdescribed above. Plasmids from the transformants may then be prepared,analyzed by restriction endonuclease digestion, and/or sequenced toconfirm the presence of the desired construct.

[0132] Vectors that provide for the transient expression of DNA encodingGDNFR in mammalian cells may also be used. In general, transientexpression involves the use of an expression vector that is able toreplicate efficiently in a host cell, such that the host cellaccumulates many copies of the expression vector and, in turn,synthesizes high levels of the desired protein encoded by the expressionvector. Transient expression systems, comprising a suitable expressionvector and a host cell, allow for the convenient positive identificationof proteins encoded by cloned DNAs, as well as for the rapid screeningof such proteins for desired biological or physiological properties.Thus, transient expression systems are particularly useful inidentifying variants of the protein.

[0133] Selection and Transformation of Host Cells

[0134] Host cells (e.g., bacterial, mammalian, insect, yeast, or plantcells) transformed with nucleic acid sequences for use in expressing arecombinant GDNFR protein are also provided by the present invention.The transformed host cell is cultured under appropriate conditionspermitting the expression of the nucleic acid sequence. The selection ofsuitable host cells and methods for transformation, culture,amplification, screening and product production and purification arewell known in the art. See for example, Gething and Sambrook, Nature,293: 620-625 (1981), or alternatively, Kaufman et al., Mol. Cell. Biol.,5 (7): 1750-1759 (1985) or Howley et al., U.S. Pat. No. 4,419,446.Additional exemplary materials and methods are discussed herein. Thetransformed host cell is cultured in a suitable medium, and theexpressed GDNFR protein is then optionally recovered, isolated andpurified from the culture medium (or from the cell, if expressedintracellularly) by an appropriate means known to those skilled in theart.

[0135] Different host cells have characteristic and specific mechanismsfor the translational and post-translational processing and modification(e.g., glycosylation, cleavage) of proteins. Appropriate cell lines orhost systems can be chosen to ensure the desired modification andprocessing of the foreign protein expressed. For example, expression ina bacterial system can be used to produce an unglycosylated core proteinproduct. Expression in yeast may be used to produce a glycosylatedproduct. Expression in mammalian cells can be used to ensure “native”glycosylation of the heterologous GDNFR protein. Furthermore, differentvector/host expression systems may effect processing reactions such asproteolytic cleavages to different extents.

[0136] Suitable host cells for cloning or expressing the vectorsdisclosed herein are prokaryote, yeast, or higher eukaryote cells.Eukaryotic microbes such as filamentous fungi or yeast may be suitablehosts for the expression of GDNFR proteins. Saccharomyces cerevisiae, orcommon baker's yeast, is the most commonly used among lower eukaryotichost microorganisms, but a number of other genera, species, and strainsare well known and commonly available.

[0137] Host cells to be used for the expression of glycosylated GDNFRprotein are also derived from multicellular organisms. Such host cellsare capable of complex processing and glycosylation activities. Inprinciple, any higher eukaryotic cell culture might be used, whethersuch culture involves vertebrate or invertebrate cells, including plantand insect cells. The propagation of vertebrate cells in culture (tissueculture) is a well known procedure. Examples of useful mammalian hostcell lines include, but are not limited to, monkey kidney CV1 linetransformed by SV40 (COS7), human embryonic kidney line (293 or 293cells subcloned for growth in suspension culture), baby hamster kidneycells, and Chinese hamster ovary cells. Other suitable mammalian celllines include but are not limited to, HeLa, mouse L-929 cells, 3T3 linesderived from Swiss, Balb-c or NIH mice, BHK or HaK hamster cell lines.

[0138] Suitable host cells also include prokaryotic cells. Prokaryotichost cells include, but are not limited to, bacterial cells, such asGram-negative or Gram-positive organisms, for example, E. coli, Bacillisuch as B. subtilis, Pseudomonas species such as P. aeruginosa,Salmonella typhimurium, or Serratia marcescans. For example, the variousstrains of E. coli (e.g., H101, DH5a, DH10, and MC1061) are well-knownas host cells in the field of biotechnology. Various strains ofStreptomyces spp. and the like may also be employed. Presently preferredhost cells for producing GDNFR proteins are bacterial cells (e.g.,Escherichia coli) and mammalian cells (such as Chinese hamster ovarycells, COS cells, etc.)

[0139] The host cells are transfected and preferably transformed withthe above-described expression or cloning vectors and cultured in aconventional nutrient medium. The medium may be modified as appropriatefor inducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences. Transfection and transformation areperformed using standard techniques which are well known to thoseskilled in the art and which are selected as appropriate to the hostcell involved. For example, for mammalian cells without cell walls, thecalcium phosphate precipitation method may be used. Electroporation,micro injection and other known techniques may also be used.

[0140] Culturing the Host Cells

[0141] Transformed cells used to produce GDNFR proteins of the presentinvention are cultured in suitable media. The media may be supplementedas necessary with hormones and/or other growth factors (such as insulin,transferrin, or epidermal growth factor), salts (such as sodiumchloride, calcium, magnesium, and phosphate), buffers (such as HEPES),nucleosides (such as adenosine and thymidine), antibiotics (such asgentamicin), trace elements (defined as inorganic compounds usuallypresent at final concentrations in the micromolar range), and glucose orother energy source. Other supplements may also be included, atappropriate concentrations, as will be appreciated by those skilled inthe art. Suitable culture conditions, such as temperature, pH, and thelike, are also well known to those skilled in the art for use with theselected host cells.

[0142] Once the GDNFR protein is produced, it may be isolated andpurified by standard methods including chromatography (e.g., ionexchange, affinity, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins. In particular, GDNFR protein may be isolatedby binding to an affinity column comprising GDNF or anti-GDNFR antibodybound to a stationary support.

[0143] Homologous Recombination

[0144] It is further envisioned that GDNFR proteins may be produced byhomologous recombination, or with recombinant production methodsutilizing control elements introduced into cells already containing DNAencoding GDNFR. For example, homologous recombination methods may beused to modify a cell that contains a normally transcriptionally silentGDNFR gene or under expressed gene and thereby produce a cell whichexpresses GDNFR. Homologous recombination is a technique originallydeveloped for targeting genes to induce or correct mutations intranscriptionally active genes (Kucherlapati, Prog. in Nucl. Acid Res.and Mol. Biol., 36:301, 1989). The basic technique was developed as amethod for introducing specific mutations into specific regions of themammalian genome (Thomas et al., Cell, 44:419-428, 1986; Thomas andCapecchi, Cell, 51:503-512, 1987; Doetschman et al., Proc. Natl. Acad.Sci., 85:8583-8587, 1988) or to correct specific mutations withindefective genes (Doetschman et al., Nature, 330:576-578, 1987).Exemplary homologous recombination techniques are described in U.S. Pat.No. 5,272,071 (EP 91 90 3051, EP Publication No. 505 500;PCT/US90/07642, International Publication No. WO 91/09955) thedisclosure of which is hereby incorporated by reference.

[0145] Through homologous recombination, the DNA sequence to be insertedinto the genome can be directed to a specific region of the gene ofinterest by attaching it to targeting DNA. The targeting DNA is DNA thatis complementary (homologous) to a region of the genomic DNA. Smallpieces of targeting DNA that are complementary to a specific region ofthe genome are put in contact with the parental strand during the DNAreplication process. It is a general property of DNA that has beeninserted into a cell to hybridize, and therefore, recombine with otherpieces of endogenous DNA through shared homologous regions. If thiscomplementary strand is attached to an oligonucleotide that contains amutation or a different sequence of DNA, it too is incorporated into thenewly synthesized strand as a result of the recombination. As a resultof the proofreading function, it is possible for the new sequence of DNAto serve as the template. Thus, the transferred DNA is incorporated intothe genome.

[0146] If the sequence of a particular gene is known, such as thenucleic acid sequence, the pre-pro sequence or expression controlsequence of GDNFR presented herein, a piece of DNA that is complementaryto a selected region of the gene can be synthesized or otherwiseobtained, such as by appropriate restriction of the native DNA atspecific recognition sites bounding the region of interest. This pieceserves as a targeting sequence upon insertion into the cell and willhybridize to its homologous region within the genome. If thishybridization occurs during DNA replication, this piece of DNA, and anyadditional sequence attached thereto, will act as an Okazaki fragmentand will be backstitched into the newly synthesized daughter strand ofDNA.

[0147] Attached to these pieces of targeting DNA are regions of DNAwhich may interact with the expression of a GDNFR protein. For example,a promoter/enhancer element, a suppresser, or an exogenous transcriptionmodulatory element is inserted in the genome of the intended host cellin proximity and orientation sufficient to influence the transcriptionof DNA encoding the desired GDNFR protein. The control element does notencode GDNFR, but instead controls a portion of the DNA present in thehost cell genome. Thus, the expression of GDNFR proteins may be achievednot by transfection of DNA that encodes the GDNFR gene itself, butrather by the use of targeting DNA (containing regions of homology withthe endogenous gene of interest) coupled with DNA regulatory segmentsthat provide the endogenous gene sequence with recognizable signals fortranscription of a GDNFR protein.

[0148] A. GDNFR variants

[0149] As discussed above, the terms “GDNFR analogs” as used hereininclude polypeptides in which amino acids have been deleted from(“deletion variants”), inserted into (“addition variants”), orsubstituted for (“substitution variants”) residues within the amino acidsequence of naturally-occurring GDNFR polypeptides including thosedepicted in FIGS. 2 and 4 (SEQ. ID. NOs.:2 and 4). Such variants areprepared by introducing appropriate nucleotide changes into the DNAencoding the polypeptide or by in vitro chemical synthesis of thedesired polypeptide. It will be appreciated by those skilled in the artthat many combinations of deletions, insertions, and substitutions canbe made to an amino acid sequence such as mature human GDNFR providedthat the final molecule possesses GDNFR activity.

[0150] Based upon the present description of GDNFR amino acid sequences,one can readily design and manufacture a variety of nucleic acidsequences suitable for use in the recombinant (e.g., microbial)expression of polypeptides having primary conformations which differfrom those depicted in the Figures in terms of the identity or locationof one or more residues. Mutagenesis techniques for the replacement,insertion or deletion of one or more selected amino acid residuesencoded by the nucleic acid sequences depicted in FIGS. 2 and 4 are wellknown to one skilled in the art (e.g., U.S. Pat. No. 4,518,584, thedisclosure of which is hereby incorporated by reference.) There are twoprincipal variables in the construction of substitution variants: thelocation of the mutation site and the nature of the mutation. Indesigning GDNFR substitution variants, the selection of the mutationsite and nature of the mutation will depend on the GDNFRcharacteristic(s) to be modified. The sites for mutation can be modifiedindividually or in series, e.g., by (1) substituting first withconservative amino acid modifications and then with more radicalselections depending upon the results achieved, (2) deleting the targetamino acid residue, or (3) inserting amino acid residues adjacent to thelocated site. Conservative changes in from 1 to 30 contiguous aminoacids are preferred. N-terminal and C-terminal deletion GDNFR proteinvariants may also be generated by proteolytic enzymes.

[0151] For GDNFR deletion variants, deletions generally range from about1 to 30 contiguous residues, more usually from about 1 to 10 contiguousresidues, and typically from about 1 to 5 contiguous residues.N-terminal, C-terminal and internal intrasequence deletions arecontemplated. Deletions may be introduced into regions of the moleculewhich have low homology with non-human GDNFR to modify the activity ofGDNFR. Deletions in areas of substantial homology with non-human GDNFRsequences will be more likely to significantly modify GDNFR biologicalactivity. The number of consecutive deletions typically will be selectedso as to preserve the tertiary structure of the GDNFR protein product inthe affected domain, e.g., cysteine crosslinking. Non-limiting examplesof deletion variants include truncated GDNFR protein products lackingN-terminal or C-terminal amino acid residues. For example, one mayprepare a soluble receptor by elimination of the peptide region involvedin a glycosyl-phosphatidylinositol (GPI) anchorage of GDNFR receptor tothe cytoplasmic membrane.

[0152] For GDNFR addition variants, amino acid sequence additionstypically include N-and/or C-terminal fusions or terminal additionsranging in length from one residue to polypeptides containing a hundredor more residues, as well as internal or medial additions of single ormultiple amino acid residues. Polypeptides of the invention may alsoinclude an initial methionine amino acid residue (at position −1 withrespect to the first amino acid residue of the desired polypeptide).Internal additions may range generally from about 1 to 10 contiguousresidues, more typically from about 1 to 5 residues, and usually fromabout 1 to 3 amino acid residues. Examples of N-terminal additionvariants include GDNFR with the inclusion of a heterologous N-terminalsignal sequence to the N-terminus of GDNFR to facilitate the secretionof mature GDNFR from recombinant host cells and thereby facilitateharvesting or bioavailability. Such signal sequences generally will beobtained from, and thus be homologous to, the intended host cellspecies. Additions may also include amino acid sequences derived fromthe sequence of other neurotrophic factors. For example, it iscontemplated that a fusion protein of GDNF and GDNFR may be produced,with or without a linking sequence, thereby forming a single moleculetherapeutic entity.

[0153] GDNFR substitution variants have one or more amino acid residuesof the GDNFR amino acid sequence removed and a different residue(s)inserted in its place. Such substitution variants include allelicvariants, which are characterized by naturally-occurring nucleotidesequence changes in the species population that may or may not result inan amino acid change. As with the other variant forms, substitutionvariants may involve the replacement of single or contiguous amino acidresidues at one or more different locations.

[0154] Specific mutations of the GDNFR amino acid sequence may involvemodifications to a glycosylation site (e.g., serine, threonine, orasparagine). The absence of glycosylation or only partial glycosylationresults from amino acid substitution or deletion at anyasparagine-linked glycosylation recognition site or at any site of themolecule that is modified by addition of an O-linked carbohydrate. Anasparagine-linked glycosylation recognition site comprises a tripeptidesequence which is specifically recognized by appropriate cellularglycosylation enzymes. These tripeptide sequences are either Asn-Xaa-Thror Asn-Xaa-Ser, where Xaa can be any amino acid other than Pro. Avariety of amino acid substitutions or deletions at one or both of thefirst or third amino acid positions of a glycosylation recognition site(and/or amino acid deletion at the second position) result innon-glycosylation at the modified tripeptide sequence. Thus, theexpression of appropriate altered nucleotide sequences produces variantswhich are not glycosylated at that site. Alternatively, the GDNFR aminoacid sequence may be modified to add glycosylation sites.

[0155] One method for identifying GDNFR amino acid residues or regionsfor mutagenesis is called “alanine scanning mutagenesis” as described byCunningham and Wells (Science, 244: 1081-1085, 1989). In this method, anamino acid residue or group of target residues are identified (e.g.,charged residues such as Arg, Asp, His, Lys, and Glu) and replaced by aneutral or negatively charged amino acid (most preferably alanine orpolyalanine) to affect the interaction of the amino acids with thesurrounding aqueous environment in or outside the cell. Those domainsdemonstrating functional sensitivity to the substitutions may then berefined by introducing additional or alternate residues at the sites ofsubstitution. Thus, the target site for introducing an amino acidsequence variation is determined, alanine scanning or random mutagenesisis conducted on the corresponding target codon or region of the DNAsequence, and the expressed GDNFR variants are screened for the optimalcombination of desired activity and degree of activity.

[0156] The sites of greatest interest for substitutional mutagenesisinclude sites where the amino acids found in GDNFR proteins from variousspecies are substantially different in terms of side-chain bulk, charge,and/or hydrophobicity. Other sites of interest are those in whichparticular residues of GDNFR-like proteins, obtained from variousspecies, are identical. Such positions are generally important for thebiological activity of a protein. Initially, these sites are substitutedin a relatively conservative manner. Such conservative substitutions areshown in Table 2 under the heading of preferred substitutions. If suchsubstitutions result in a change in biological activity, then moresubstantial changes (exemplary substitutions) may be introduced, and/orother additions or deletions may be made, and the resulting products arescreened for activity. TABLE 2 Amino Acid Substitutions Original ResiduePreferred Substitutions Exemplary Substitutions Ala (A) Val Val; Leu;Ile Arg (R) Lys Lys; Gln; Asn Asn (N) Gln Gln; His; Lys; Arg Asp (D) GluGlu Cys (C) Ser Ser Gln (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro Pro His(H) Arg Asn; Gln; Lys; Arg Ile (I) Leu Leu; Val; Met; Ala; Phe;norleucine Leu (L) Ile norleucine; Ile; Val; Met; Ala; Phe Lys (K) ArgArg; Gln; Asn Met (M) Leu Leu; Phe; Ile Phe (F) Leu Leu; Val; Ile; AlaPro (P) Gly Gly Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr Tyr Tyr (Y)Phe Trp; Phe; Thr; Ser Val (V) Leu Ile; Leu; Met; Phe; Ala; norleucine

[0157] Conservative modifications to the amino acid sequence (and thecorresponding modifications to the encoding nucleic acid sequences) areexpected to produce GDNFR protein products having functional andchemical characteristics similar to those of naturally occurring GDNFR.In contrast, substantial modifications in the functional and/or chemicalcharacteristics of GDNFR protein products may be accomplished byselecting substitutions that differ significantly in their effect onmaintaining (a) the structure of the polypeptide backbone in the area ofthe substitution, for example, as a sheet or helical conformation, (b)the charge or hydrophobicity of the molecule at the target site, or (c)the bulk of the side chain. Naturally occurring residues may be dividedinto groups based on common side chain properties:

[0158] 1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

[0159] 2) neutral hydrophilic: Cys, Ser, Thr;

[0160] 3) acidic: Asp, Glu;

[0161] 4) basic: Asn, Gln, His, Lys, Arg;

[0162] 5) residues that influence chain orientation: Gly, Pro; and

[0163] 6) aromatic: Trp, Tyr, Phe.

[0164] Non-conservative substitutions may involve the exchange of amember of one of these classes for a member from another class. Suchsubstituted residues may be introduced into regions of the human GDNFRprotein that are homologous with non-human GDNFR proteins, or into thenon-homologous regions of the molecule.

[0165] Thus, GDNFR proteins, analogs, or derivatives thereof include,but are not limited to, those biologically active molecules containing,as a primary amino acid sequence, all or part of the amino acidsequences as depicted in FIGS. 2 and 4 (SEQ ID NOs. 2 and 4). Theproteins will include altered sequences in which biologically equivalentamino acid residues are substituted for residues within the sequenceresulting in a silent change. For example, one or more amino acidresidues within the sequence can be substituted by another amino acid ofa similar polarity which acts as a functional equivalent, resulting in asilent alteration. Substitutes for an amino acid within the sequence maybe selected from other members of the class to which the amino acidbelongs. For example, the nonpolar (hydrophobic) amino acids includealanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophanand methionine. The polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine. The positivelycharged (basic) amino acids include arginine, lysine and histidine. Thenegatively charged (acidic) amino acids include aspartic acid andglutamic acid. It is also contemplated that the GDNFR proteins, analogs,or fragments or derivatives thereof may be differentially modifiedduring or after translation, e.g., by phosphorylation, glycosylation,crosslinking, acylation, proteolytic cleavage, linkage to an antibodymolecule, membrane molecule or other ligand.

[0166] B. GDNFR Derivatives

[0167] Chemically modified derivatives of GDNFR or GDNFR analogs may beprepared by one of skill in the art based upon the present disclosure.The chemical moieties most suitable for derivatization include watersoluble polymers. A water soluble polymer is desirable because theprotein to which it is attached does not precipitate in an aqueousenvironment, such as a physiological environment. Preferably, thepolymer will be pharmaceutically acceptable for the preparation of atherapeutic product or composition. One skilled in the art will be ableto select the desired polymer based on such considerations as whetherthe polymer/protein conjugate will be used therapeutically, and if so,the desired dosage, circulation time, resistance to proteolysis, andother considerations. The effectiveness of the derivatization may beascertained by administering the derivative, in the desired form (e.g.,by osmotic pump, or, more preferably, by injection or infusion, or,further formulated for oral, pulmonary or other delivery routes), anddetermining its effectiveness.

[0168] Suitable water soluble polymers include, but are not limited to,polyethylene glycol, copolymers of ethylene glycol/propylene glycol,carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleicanhydride copolymer, polyaminoacids (either homopolymers or randomcopolymers), and dextran or poly(n-vinyl pyrrolidone)polyethyleneglycol, propropylene glycol homopolymers, prolypropylene oxide/ethyleneoxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinylalcohol, and mixtures thereof. Polyethylene glycol propionaldehyde mayhave advantages in manufacturing due to its stability in water.

[0169] The polymer may be of any molecular weight, and may be branchedor unbranched. For polyethylene glycol, the preferred molecular weightis between about 2 kDa and about 100 kDa for ease in handling andmanufacturing (the term “about” indicating that in preparations ofpolyethylene glycol, some molecules will weigh more, some less, than thestated molecular weight). Other sizes may be used, depending on thedesired therapeutic profile (e.g., the duration of sustained releasedesired; the effects, if any, on biological activity; the ease inhandling; the degree or lack of antigenicity and other known effects ofpolyethylene glycol on a therapeutic protein or variant).

[0170] The number of polymer molecules so attached may vary, and oneskilled in the art will be able to ascertain the effect on function. Onemay mono-derivatize, or may provide for a di-, tri-, tetra- or somecombination of derivatization, with the same or different chemicalmoieties (e.g., polymers, such as different weights of polyethyleneglycols). The proportion of polymer molecules to protein (or peptide)molecules will vary, as will their concentrations in the reactionmixture. In general, the optimum ratio (in terms of efficiency ofreaction in that there is no excess unreacted protein or polymer) willbe determined by factors such as the desired degree of derivatization(e.g., mono, di-, tri-, etc.), the molecular weight of the polymerselected, whether the polymer is branched or unbranched, and thereaction conditions.

[0171] The polyethylene glycol molecules (or other chemical moieties)should be attached to the protein with consideration of effects onfunctional or antigenic domains of the protein. There are a number ofattachment methods available to those skilled in the art. See forexample, EP 0 401 384, the disclosure of which is hereby incorporated byreference (coupling PEG to G-CSF), see also Malik et al., Exp. Hematol.,20: 1028-1035, 1992 (reporting pegylation of GM-CSF using tresylchloride). For example, polyethylene glycol may be covalently boundthrough amino acid residues via a reactive group, such as, a free aminoor carboxyl group. Reactive groups are those to which an activatedpolyethylene glycol molecule may be bound. The amino acid residueshaving a free amino group may include lysine residues and the N-terminalamino acid residue. Those having a free carboxyl group may includeaspartic acid residues, glutamic acid residues, and the C-terminal aminoacid residue. Sulfhydrl groups may also be used as a reactive group forattaching the polyethylene glycol molecule(s). For therapeutic purposes,attachment at an amino group, such as attachment at the N-terminus orlysine group is preferred. Attachment at residues important for receptorbinding should be avoided if receptor binding is desired.

[0172] One may specifically desire an N-terminal chemically modifiedprotein. Using polyethylene glycol as an illustration of the presentcompositions, one may select from a variety of polyethylene glycolmolecules (by molecular weight, branching, etc.), the proportion ofpolyethylene glycol molecules to protein (or peptide) molecules in thereaction mix, the type of pegylation reaction to be performed, and themethod of obtaining the selected N-terminally pegylated protein. Themethod of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)may be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective N-terminal chemicalmodification may be accomplished by reductive alkylation which exploitsdifferential reactivity of different types of primary amino groups(lysine versus the N-terminal) available for derivatization in aparticular protein. Under the appropriate reaction conditions,substantially selective derivatization of the protein at the N-terminuswith a carbonyl group containing polymer is achieved. For example, onemay selectively N-terminally pegylate the protein by performing thereaction at a pH which allows one to take advantage of the pKadifferences between the e-amino group of the lysine residues and that ofthe a-amino group of the N-terminal residue of the protein. By suchselective derivatization, attachment of a water soluble polymer to aprotein is controlled: the conjugation with the polymer takes placepredominantly at the N-terminus of the protein and no significantmodification of other reactive groups, such as the lysine side chainamino groups, occurs. Using reductive alkylation, the water solublepolymer may be of the type described above, and should have a singlereactive aldehyde for coupling to the protein. Polyethylene glycolpropionaldehyde, containing a single reactive aldehyde, may be used.

[0173] The present invention contemplates use of derivatives which areprokaryote-expressed GDNFR, or variants thereof, linked to at least onepolyethylene glycol molecule, as well as use of GDNFR, or variantsthereof, attached to one or more polyethylene glycol molecules via anacyl or alkyl linkage.

[0174] Pegylation may be carried out by any of the pegylation reactionsknown in the art. See, for example: Focus on Growth Factors, 3 (2):4-10, 1992; EP 0 154 316, the disclosure of which is hereby incorporatedby reference; EP 0 401 384; and the other publications cited herein thatrelate to pegylation. The pegylation may be carried out via an acylationreaction or an alkylation reaction with a reactive polyethylene glycolmolecule (or an analogous reactive water-soluble polymer).

[0175] Pegylation by acylation generally involves reacting an activeester derivative of polyethylene glycol (PEG) with the GDNFR protein orvariant. Any known or subsequently discovered reactive PEG molecule maybe used to carry out the pegylation of GDNFR protein or variant. Apreferred activated PEG ester is PEG esterified to N-hydroxysuccinimide(NHS). As used herein, “acylation” is contemplated to include withoutlimitation the following types of linkages between the therapeuticprotein and a water soluble polymer such as PEG: amide, carbamate,urethane, and the like. See Bioconjugate Chem., 5: 133-140, 1994.Reaction conditions may be selected from any of those known in thepegylation art or those subsequently developed, but should avoidconditions such as temperature, solvent, and pH that would inactivatethe GDNFR or variant to be modified.

[0176] Pegylation by acylation will generally result in a poly-pegylatedGDNFR protein or variant. Preferably, the connecting linkage will be anamide. Also preferably, the resulting product will be substantially only(e.g., >95%) mono, di- or tri-pegylated. However, some species withhigher degrees of pegylation may be formed in amounts depending on thespecific reaction conditions used. If desired, more purified pegylatedspecies may be separated from the mixture, particularly unreactedspecies, by standard purification techniques, including, among others,dialysis, salting-out, ultrafiltration, ion-exchange chromatography, gelfiltration chromatography and electrophoresis.

[0177] Pegylation by alkylation generally involves reacting a terminalaldehyde derivative of PEG with the GDNFR protein or variant in thepresence of a reducing agent. Pegylation by alkylation can also resultin poly-pegylated GDNFR protein or variant. In addition, one canmanipulate the reaction conditions to favor pegylation substantiallyonly at the a-amino group of the N-terminus of the GDNFR protein orvariant (i.e., a mono-pegylated protein). In either case ofmonopegylation or polypegylation, the PEG groups are preferably attachedto the protein via a —CH2—NH— group. With particular reference to the—CH2— group, this type of linkage is referred to herein as an “alkyl”linkage.

[0178] Derivatization via reductive alkylation to produce amonopegylated product exploits differential reactivity of differenttypes of primary amino groups (lysine versus the N-terminal) availablefor derivatization. The reaction is performed at a pH which allows oneto take advantage of the pKa differences between the e-amino groups ofthe lysine residues and that of the a-amino group of the N-terminalresidue of the protein. By such selective derivatization, attachment ofa water soluble polymer that contains a reactive group such as analdehyde, to a protein is controlled: the conjugation with the polymertakes place predominantly at the N-terminus of the protein and nosignificant modification of other reactive groups, such as the lysineside chain amino groups, occurs. In one important aspect, the presentinvention contemplates use of a substantially homogeneous preparation ofmonopolymer/GDNFR protein (or variant) conjugate molecules (meaning GDNFprotein or variant to which a polymer molecule has been attachedsubstantially only (i.e., >95%) in a single location). Morespecifically, if polyethylene glycol is used, the present invention alsoencompasses use of pegylated GDNFR protein or variant lacking possiblyantigenic linking groups, and having the polyethylene glycol moleculedirectly coupled to the GDNFR protein or variant.

[0179] Thus, GDNFR protein products according to the present inventioninclude pegylated GDNFR protein or variants, wherein the PEG group(s) is(are) attached via acyl or alkyl groups. As discussed above, suchproducts may be mono-pegylated or poly-pegylated (e.g., containing 2-6,and preferably 2-5, PEG groups). The PEG groups are generally attachedto the protein at the a- or e-amino groups of amino acids, but it isalso contemplated that the PEG groups could be attached to any aminogroup attached to the protein, which is sufficiently reactive to becomeattached to a PEG group under suitable reaction conditions.

[0180] The polymer molecules used in both the acylation and alkylationapproaches may be selected from among water soluble polymers asdescribed above. The polymer selected should be modified to have asingle reactive group, such as an active ester for acylation or analdehyde for alkylation, preferably, so that the degree ofpolymerization may be controlled as provided for in the present methods.An exemplary reactive PEG aldehyde is polyethylene glycolpropionaldehyde, which is water stable, or mono C1-C10 alkoxy or aryloxyderivatives thereof (see, U.S. Pat. No. 5,252,714). The polymer may bebranched or unbranched. For the acylation reactions, the polymer(s)selected should have a single reactive ester group. For the presentreductive alkylation, the polymer(s) selected should have a singlereactive aldehyde group. Generally, the water soluble polymer will notbe selected from naturally-occurring glycosyl residues since these areusually made more conveniently by mammalian recombinant expressionsystems. The polymer may be of any molecular weight, and may be branchedor unbranched.

[0181] An exemplary water-soluble polymer for use herein is polyethyleneglycol. As used herein, polyethylene glycol is meant to encompass any ofthe forms of PEG that have been used to derivatize other proteins, suchas mono-(C1-C10) alkoxy- or aryloxy-polyethylene glycol.

[0182] In general, chemical derivatization may be performed under anysuitable condition used to react a biologically active substance with anactivated polymer molecule. Methods for preparing a pegylated GDNFRprotein product will generally comprise the steps of (a) reacting aGDNFR protein product with polyethylene glycol (such as a reactive esteror aldehyde derivative of PEG) under conditions whereby the proteinbecomes attached to one or more PEG groups, and (b) obtaining thereaction product(s). In general, the optimal reaction conditions for theacylation reactions will be determined case-by-case based on knownparameters and the desired result. For example, the larger the ratio ofPEG:protein, the greater the percentage of poly-pegylated product.

[0183] Reductive alkylation to produce a substantially homogeneouspopulation of mono-polymer/GDNFR protein product will generally comprisethe steps of: (a) reacting a GDNFR protein or variant with a reactivePEG molecule under reductive alkylation conditions, at a pH suitable topermit selective modification of the a-amino group at the amino terminusof said GDNFR protein or variant; and (b) obtaining the reactionproduct(s).

[0184] For a substantially homogeneous population of mono-polymer/GDNFRprotein product, the reductive alkylation reaction conditions are thosewhich permit the selective attachment of the water soluble polymermoiety to the N-terminus of GDNFR protein or variant. Such reactionconditions generally provide for pKa differences between the lysineamino groups and the a-amino group at the N-terminus (the pKa being thepH at which 50% of the amino groups are protonated and 50% are not). ThepH also affects the ratio of polymer to protein to be used. In general,if the pH is lower, a larger excess of polymer to protein will bedesired (i.e., the less reactive the N-terminal a-amino group, the morepolymer needed to achieve optimal conditions). If the pH is higher, thepolymer:protein ratio need not be as large (i.e., more reactive groupsare available, so fewer polymer molecules are needed). For purposes ofthe present invention, the pH will generally fall within the range of3-9, preferably 3-6.

[0185] Another important consideration is the molecular weight of thepolymer. In general, the higher the molecular weight of the polymer, thefewer polymer molecules may be attached to the protein. Similarly,branching of the polymer should be taken into account when optimizingthese parameters. Generally, the higher the molecular weight (or themore branches) the higher the polymer:protein ratio. In general, for thepegylation reactions contemplated herein, the preferred averagemolecular weight is about 2 kDa to about 100 kDa. The preferred averagemolecular weight is about 5 kDa to about 50 kDa, particularly preferablyabout 12 kDa to about 25 kDa. The ratio of water-soluble polymer to GDNFprotein or variant will generally range from 1:1 to 100:1, preferably(for polypegylation) 1:1 to 20:1 and (for monopegylation) 1:1 to 5:1.

[0186] Using the conditions indicated above, reductive alkylation willprovide for selective attachment of the polymer to any GDNFR protein orvariant having an a-amino group at the amino terminus, and provide for asubstantially homogenous preparation of monopolymer/GDNFR protein (orvariant) conjugate. The term “monopolymer/GDNFR protein (or variant)conjugate” is used here to mean a composition comprised of a singlepolymer molecule attached to a molecule of GDNFR protein or GDNFRvariant protein. The monopolymer/GDNFR protein (or variant) conjugatetypically will have a polymer molecule located at the N-terminus, butnot on lysine amino side groups. The preparation will generally begreater than 90% monopolymer/GDNFR protein (or variant) conjugate, andmore usually greater than 95% monopolymer/GDNFR protein (or variant)conjugate, with the remainder of observable molecules being unreacted(i.e., protein lacking the polymer moiety). It is also envisioned thatthe GDNFR protein product may involve the preparation of a pegylatedmolecule involving a fusion protein or linked GDNFR and GDNF molecules.

[0187] For the present reductive alkylation, the reducing agent shouldbe stable in aqueous solution and preferably be able to reduce only theSchiff base formed in the initial process of reductive alkylation.Suitable reducing agents may be selected from sodium borohydride, sodiumcyanoborohydride, dimethylamine borane, trimethylamine borane andpyridine borane. A particularly suitable reducing agent is sodiumcyanoborohydride. Other reaction parameters, such as solvent, reactiontimes, temperatures, etc., and means of purification of products, can bedetermined case-by-case based on the published information relating toderivatization of proteins with water soluble polymers (see thepublications cited herein).

[0188] C. GDNFR Protein Product Pharmaceutical Compositions

[0189] GDNFR protein product pharmaceutical compositions typicallyinclude a therapeutically or prophylactically effective amount of GDNFRprotein product in admixture with one or more pharmaceutically andphysiologically acceptable formulation materials selected forsuitability with the mode of administration. Suitable formulationmaterials include, but are not limited to, antioxidants, preservatives,coloring, flavoring and diluting agents, emulsifying agents, suspendingagents, solvents, fillers, bulking agents, buffers, delivery vehicles,diluents, excipients and/or pharmaceutical adjuvants. For example, asuitable vehicle may be water for injection, physiological salinesolution, or artificial cerebrospinal fluid, possibly supplemented withother materials common in compositions for parenteral administration.Neutral buffered saline or saline mixed with serum albumin are furtherexemplary vehicles. The term “pharmaceutically acceptable carrier” or“physiologically acceptable carrier” as used herein refers to aformulation material(s) suitable for accomplishing or enhancing thedelivery of the GDNFR protein product as a pharmaceutical composition.

[0190] The primary solvent in a vehicle may be either aqueous ornon-aqueous in nature. In addition, the vehicle may contain otherformulation materials for modifying or maintaining the pH, osmolarity,viscosity, clarity, color, sterility, stability, rate of dissolution, orodor of the formulation. Similarly, the vehicle may contain additionalformulation materials for modifying or maintaining the rate of releaseof GDNFR protein product, or for promoting the absorption or penetrationof GDNFR protein product across the blood-brain barrier.

[0191] Once the therapeutic pharmaceutical composition has beenformulated, it may be stored in sterile vials as a solution, suspension,gel, emulsion, solid, or dehydrated or lyophilized powder. Suchformulations may be stored either in a ready to use form or in a form(e.g., lyophilized) requiring reconstitution prior to administration.

[0192] The optimal pharmaceutical formulation will be determined by oneskilled in the art depending upon the intended route of administrationand desired dosage. See for example, Remington's PharmaceuticalSciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pages1435-1712, the disclosure of which is hereby incorporated by reference.Such compositions may influence the physical state, stability, rate ofin vivo release, and rate of in vivo clearance of the present proteinsand derivatives.

[0193] Effective administration forms, such as (1) slow-releaseformulations, (2) inhalant mists, or (3) orally active formulations areenvisioned. The GDNFR protein product pharmaceutical composition alsomay be formulated for parenteral administration. Such parenterallyadministered therapeutic compositions are typically in the form of apyrogen-free, parenterally acceptable aqueous solution comprising theGDNFR protein product in a pharmaceutically acceptable vehicle. Onepreferred vehicle is physiological saline. The GDNFR protein productpharmaceutical compositions also may include particulate preparations ofpolymeric compounds such as polylactic acid, polyglycolic acid, etc. orinto liposomes. Hyaluronic acid may also be used, and this may have theeffect of promoting sustained duration in the circulation.

[0194] A particularly suitable vehicle for parenteral injection issterile distilled water in which the GDNFR protein product is formulatedas a sterile, isotonic solution, properly preserved. Yet anotherpreparation may involve the formulation of the GDNFR protein productwith an agent, such as injectable microspheres or liposomes, thatprovides for the slow or sustained release of the protein which may thenbe delivered as a depot injection. Other suitable means for theintroduction of GDNFR protein product include implantable drug deliverydevices which contain the GDNFR protein product.

[0195] The preparations of the present invention may include othercomponents, for example parenterally acceptable preservatives, tonicityagents, cosolvents, wetting agents, complexing agents, buffering agents,antimicrobials, antioxidants and surfactants, as are well known in theart. For example, suitable tonicity enhancing agents include alkalimetal halides (preferably sodium or potassium chloride), mannitol,sorbitol and the like. Suitable preservatives include, but are notlimited to, benzalkonium chloride, thimerosal, phenethyl alcohol,methylparaben, propylparaben, chlorhexidine, sorbic acid and the like.Hydrogen peroxide may also be used as preservative. Suitable cosolventsare for example glycerin, propylene glycol and polyethylene glycol.Suitable complexing agents are for example caffeine,polyvinylpyrrolidone, beta-cyclodextrin orhydroxypropyl-beta-cyclodextrin. Suitable surfactants or wetting agentsinclude sorbitan esters, polysorbates such as polysorbate 80,tromethamine, lecithin, cholesterol, tyloxapal and the like. The bufferscan be conventional buffers such as borate, citrate, phosphate,bicarbonate, or Tris-HCl.

[0196] The formulation components are present in concentration that areacceptable to the site of administration. For example, buffers are usedto maintain the composition at physiological pH or at slightly lower pH,typically within a pH range of from about 5 to about 8.

[0197] A pharmaceutical composition may be formulated for inhalation.For example, the GDNFR protein product may be formulated as a dry powderfor inhalation. GDNFR protein product inhalation solutions may also beformulated in a liquefied propellant for aerosol delivery. In yetanother formulation, solutions may be nebulized.

[0198] It is also contemplated that certain formulations containingGDNFR protein product are to be administered orally. GDNFR proteinproduct which is administered in this fashion may be formulated with orwithout those carriers customarily used in the compounding of soliddosage forms such as tablets and capsules. For example, a capsule may bedesigned to release the active portion of the formulation at the pointin the gastrointestinal tract when bioavailability is maximized andpre-systemic degradation is minimized. Additional formulation materialsmay be included to facilitate absorption of GDNFR protein product.Diluents, flavorings, low melting point waxes, vegetable oils,lubricants, suspending agents, tablet disintegrating agents, and bindersmay also be employed.

[0199] Another preparation may involve an effective quantity of GDNFRprotein product in a mixture with non-toxic excipients which aresuitable for the manufacture of tablets. By dissolving the tablets insterile water, or other appropriate vehicle, solutions can be preparedin unit dose form. Suitable excipients include, but are not limited to,inert diluents, such as calcium carbonate, sodium carbonate orbicarbonate, lactose, or calcium phosphate; or binding agents, such asstarch, gelatin, or acacia; or lubricating agents such as magnesiumstearate, stearic acid, or talc.

[0200] Additional GDNFR protein product formulations will be evident tothose skilled in the art, including formulations involving GDNFR proteinproduct in combination with GDNF protein product. Techniques forformulating a variety of other sustained- or controlled-delivery means,such as liposome carriers, bio-erodible microparticles or porous beadsand depot injections, are also known to those skilled in the art. See,for example, Supersaxo et al. description of controlled release porouspolymeric microparticles for the delivery of pharmaceutical compositions(International Publication No. WO 93/15722; International ApplicationNo. PCT/US93/00829) the disclosure of which is hereby incorporated byreference.

[0201] D. Administration of GDNFR Protein Product

[0202] The GDNFR protein product may be administered parenterally via avariety of routes, including subcutaneous, intramuscular, intravenous,transpulmonary, transdermal, intrathecal and intracerebral delivery. Inaddition, protein factors that do not readily cross the blood-brainbarrier may be given directly intracerebrally or otherwise inassociation with other elements that will transport them across thebarrier. For example, the GDNFR protein product may be administeredintracerebroventricularly or into the brain or spinal cord subarachnoidspace. GDNFR protein product may also be administered intracerebrallydirectly into the brain parenchyma. GDNFR protein product may beadministered extracerebrally in a form that has been modified chemicallyor packaged so that it passes the blood-brain barrier, or with one ormore agents capable of promoting penetration of GDNFR protein productacross the barrier. For example, a conjugate of NGF and monoclonalanti-transferrin receptor antibodies has been shown to be transported tothe brain via binding to transferrin receptors.

[0203] To achieve the desired level of GDNFR protein product, repeateddaily or less frequent injections may be administered, or GDNFR proteinproduct may be infused continuously or periodically from a constant- orprogrammable-flow implanted pump. Slow-releasing implants containing theneurotrophic factor embedded in a biodegradable polymer matrix can alsodeliver GDNFR protein product. The frequency of dosing will depend onthe pharmacokinetic parameters of the GDNFR protein product asformulated, and the route and site of administration.

[0204] Regardless of the manner of administration, the specific dose maybe calculated according to body weight, body surface area or organ size.Further refinement of the calculations necessary to determine theappropriate dosage for treatment involving each of the above mentionedformulations is routinely made by those of ordinary skill in the art andis within the ambit of tasks routinely performed by them. Appropriatedosages may be ascertained through use of appropriate dose-responsedata.

[0205] The final dosage regimen involved in a method for treating aspecific injury or condition will be determined by the attendingphysician. Generally, an effective amount of the present GDNFRpolypeptides will be determined by considering various factors whichmodify the action of drugs, e.g. the age, condition, body weight, sexand diet of the patient, the severity of any infection, time ofadministration and other clinical factors. See, Remington'sPharmaceutical Sciences, supra, at pages 697-773, herein incorporated byreference. It is contemplated that if GDNFR is used to enhance GDNFaction, then the GDNFR dose is selected to be similar to that requiredfor GDNF therapy; if GDNFR is used to antagonize GDNF action, then theGDNFR dose would be several many times the GDNF dose. Dosing may be oneor more times daily, or less frequently, and may be in conjunction withother compositions as described herein. It should be noted that thepresent invention is not limited to the dosages recited herein.

[0206] It is envisioned that the continuous administration or sustaineddelivery of GDNFR protein products may be advantageous for a giventreatment. While continuous administration may be accomplished via amechanical means, such as with an infusion pump, it is contemplated thatother modes of continuous or near continuous administration may bepracticed. For example, chemical derivatization or encapsulation mayresult in sustained release forms of the protein which have the effectof continuous presence in the bloodstream, in predictable amounts, basedon a determined dosage regimen. Thus, GDNFR protein products includeproteins derivatized or otherwise formulated to effectuate suchcontinuous administration. Sustained release forms of the GDNFR proteinproducts will be formulated to provide the desired daily or weeklyeffective dosage.

[0207] It is further contemplated that the GDNFR protein product may beadministered in a combined form with GDNF. Alternatively, the GDNFR andGDNF protein products may be administered separately, eithersequentially or simultaneously.

[0208] GDNFR protein product of the present invention may also beemployed, alone or in combination with other growth factors in thetreatment of nerve disease. In addition, other factors or othermolecules, including chemical compositions, may be employed togetherwith a GDNFR protein product. In the treatment of Parkinson's Disease,it is contemplated that GDNFR protein product be used by itself or inconjunction with the administration of Levodopa, wherein the GDNFR wouldenhance the activity of endogenous GDNF and thereby enhance the neuronaluptake of the increased concentration of dopamine.

[0209] As stated above, it is also contemplated that additionalneurotrophic or neuron nurturing factors will be useful or necessary totreat some neuronal cell populations or some types of injury or disease.Other factors that may be used in conjunction with GDNFR or acombination of GDNFR and GDNF include, but are not limited to: mitogenssuch as insulin, insulin-like growth factors, epidermal growth factor,vasoactive growth factor, pituitary adenylate cyclase activatingpolypeptide, interferon and somatostatin; neurotrophic factors such asnerve growth factor, brain derived neurotrophic factor, neurotrophin-3,neurotrophin-4/5, neurotrophin-6, insulin-like growth factor, ciliaryneurotrophic factor, acidic and basic fibroblast growth factors,fibroblast growth factor-5, transforming growth factor-β,cocaine-amphetamine regulated transcript (CART); and other growthfactors such as epidermal growth factor, leukemia inhibitory factor,interleukins, interferons, and colony stimulating factors; as well asmolecules and materials which are the functional equivalents to thesefactors.

[0210] GDNFR Protein Product Cell Therapy and Gene Therapy

[0211] GDNFR protein product cell therapy, e.g., intracerebralimplantation of cells producing GDNFR protein product, is alsocontemplated. This embodiment would involve implanting into patientscells capable of synthesizing and secreting a biologically active formof GDNFR protein product. Such GDNFR protein product-producing cells maybe cells that are natural producers of GDNFR protein product or may berecombinant cells whose ability to produce GDNFR protein product hasbeen augmented by transformation with a gene encoding the desired GDNFRprotein product. Such a modification may be accomplished by means of avector suitable for delivering the gene as well as promoting itsexpression and secretion. In order to minimize a potential immunologicalreaction in patients being administered a GDNFR protein product of aforeign species, it is preferred that the natural cells producing GDNFRprotein product be of human origin and produce human GDNFR proteinproduct. Likewise, it is preferred that the recombinant cells producingGDNFR protein product be transformed with an expression vectorcontaining a gene encoding a human GDNFR protein product.

[0212] Implanted cells may be encapsulated to avoid infiltration ofsurrounding tissue. Human or non-human animal cells may be implanted inpatients in biocompatible, semipermeable polymeric enclosures ormembranes that allow release of GDNFR protein product, but that preventdestruction of the cells by the patient's immune system or by otherdetrimental factors from the surrounding tissue. Alternatively, thepatient's own cells, transformed to produce GDNFR protein product exvivo, could be implanted directly into the patient without suchencapsulation.

[0213] Techniques for the encapsulation of living cells are familiar tothose of ordinary skill in the art, and the preparation of theencapsulated cells and their implantation in patients may beaccomplished without undue experimentation. For example, Baetge et al.(International Publication No. WO 95/05452; International ApplicationNo. PCT/US94/09299 the disclosure of which is hereby incorporated byreference) describe biocompatible capsules containing geneticallyengineered cells for the effective delivery of biologically activemolecules. In addition, see U.S. Pat, Nos. 4,892,538, 5,011,472, and5,106,627, each of which is specifically incorporated herein byreference. A system for encapsulating living cells is described in PCTApplication WO 91/10425 of Aebischer et al., specifically incorporatedherein by reference. See also, PCT Application WO 91/10470 of Aebischeret al., Winn et al., Exper. Neurol., 113:322-329, 1991, Aebischer etal., Exper. Neurol., 111:269-275, 1991; Tresco et al., ASAIO, 38:17-23,1992, each of which is specifically incorporated herein by reference.

[0214] In vivo and in vitro gene therapy delivery of GDNFR proteinproduct is also envisioned. In vitro gene therapy may be accomplished byintroducing the gene coding for GDNFR protein product into targetedcells via local injection of a nucleic acid construct or otherappropriate delivery vectors. (Hefti, J. Neurobiol,. 25:1418-1435,1994). For example, a nucleic acid sequence encoding a GDNFR proteinproduct may be contained in an adeno-associated virus vector fordelivery into the targeted cells (e.g., Johnson, InternationalPublication No. WO 95/34670; International Application No.PCT/US95/07178 the disclosure of which is hereby incorporated byreference). Alternative viral vectors include, but are not limited to,retrovirus, adenovirus, herpes simplex virus and papilloma virusvectors. Physical transfer, either in vivo or ex vivo as appropriate,may also be achieved by liposome-mediated transfer, direct injection(naked DNA), receptor-mediated transfer (ligand-DNA complex),electroporation, calcium phosphate precipitation or microparticlebombardment (gene gun).

[0215] It is also contemplated that GDNFR protein product gene therapyor cell therapy can further include the delivery of GDNF proteinproduct. For example, the host cell may be modified to express andrelease both GDNFR protein product and GDNF protein product.Alternatively, the GDNFR and GDNF protein products may be expressed inand released from separate cells. Such cells may be separatelyintroduced into the patient or the cells may be contained in a singleimplantable device, such as the encapsulating membrane described above.

[0216] It should be noted that the GDNFR protein product formulationsdescribed herein may be used for veterinary as well as humanapplications and that the term “patient” should not be construed in alimiting manner. In the case of veterinary applications, the dosageranges may be determined as described above.

EXAMPLES Example 1 Identification of Cells Expressing High Affinity GDNFBinding Sites

[0217] Expression cloning involved the selection of a source of mRNAwhich is likely to contain significant levels of the target transcript.Retina photoreceptor cells were identified as responsive to GDNF at verylow concentrations, suggesting the existence of a functional, highaffinity receptor. To confirm that rat photoreceptor cells did express ahigh affinity receptor for GDNF, [¹²⁵I]GDNF binding and photographicemulsion analysis were carried out.

[0218] Rat Retinal Cell Cultures

[0219] The neural retinas of 5-day-old C57B1/6 mouse pups or 3-day-oldSprague-Dawley rat pups (Jackson Laboratories, Bar Harbor, Mass.) werecarefully removed and dissected free of the pigment epithelium, cut into1 mm² fragments and placed into ice-cold phosphate-buffered saline(PBS). The retinas were then transferred into 10 mL of Hank's balancedsalt solution (HBSS) containing 120 units papain and 2000 units DNAaseand incubated for 20 minutes at 37° C. on a rotary platform shaker atabout 200 rpm. The cells were then dispersed by trituration throughfire-polished Pasteur pipettes, sieved through a 20 μm Nitex nylon meshand centrifuged for five minutes at 200×g . The resulting cell pelletwas resuspended into HBSS containing 1% ovalbumin and 500 units DNAase,layered on top of a 4% ovalbumin solution (in HBSS) and centrifuged for10 minutes at 500×g. The final pellet was resuspended in completeculture medium (see below), adjusted to about 15,000 cells/mL, andseeded in 90 μl aliquots into tissue culture plates coated withpolyornithine and laminin as previously described (Louis et al., JournalOf Pharmacology And Experimental Therapeutics, 262, 1274-1283, 1992).

[0220] The culture medium consisted of a 1:1 mixture of Dulbecco'sModified Eagle's Medium (DMEM) and F12 medium, and was supplemented with2.5% heat-inactivated horse serum (Hyclone, Logan, Utah), B27 mediumsupplement (GIBCO, Grand Island, N.Y.), D-glucose (final concentration:5 mg/mL), L-glutamine (final concentration: 2 mM), 20 mM HEPES, bovineinsulin and human transferrin (final concentrations: 2.5 and 0.1 mg/mL,respectively).

[0221] Immunocytochemical Identification of Photoreceptors

[0222] Photoreceptors were identified by immunostaining for arrestin, arod-specific antigen. Cultures of photoreceptors were fixed for 30minutes at room temperature with 4% paraformaldehyde in PBS, pH 7.4,followed by three washes in PBS. The fixed cultures were then incubatedin Superblock blocking buffer (Pierce, Rockford, Ill.), containing 1%Nonidet P-40 to increase the penetration of the antibodies. Theanti-arrestin antibodies (polyclonal rabbit antibody against thesynthetic peptide sequence of arrestin:Val-Phe-Glu-Glu-Phe-Ala-Arg-Gln-Asn-Leu-Lys-Cys) were then applied at adilution of between 1:2000 in the same buffer, and the cultures wereincubated for one hour at 37° C. on a rotary shaker. After three washeswith PBS, the cultures were incubated for one hour at 37° C. withgoat-anti-rabbit IgG (Vectastain kit from Vector Laboratories,Burlingame, Calif.) at a 1:500 dilution. After three washes with PBS,the secondary antibodies were then labeled with anavidin-biotin-peroxidase complex diluted at 1:500 (45 minutes at 37°C.). After three more washes with PBS, the labeled cell cultures werereacted for 5-20 minutes in a solution of 0.1 M Tris-HCl, pH 7.4,containing 0.04% 3′,3′-diaminobenzidine-(HCl)4, 0.06 percent NiCl₂ and0.02 percent hydrogen peroxide. Based on arrestin-immunoreactivity,about 90% of the cells in the cultures were rod photoreceptors.

[0223] The survival of photoreceptors was determined by examination ofarrestin-stained cultures with bright-light optics at 200×magnification. The number of arrestin-positive photoreceptors wascounted in one diametrical 1×6 mm strip, representing about 20 percentof the total surface area of a 6 mm-well. Viable photoreceptors werecharacterized as having a regularly-shaped cell body, with a usuallyshort axon-like process. Photoreceptors showing signs of degeneration,such as having irregular, vacuolated perikarya or fragmented neurites,were excluded from the counts (most of the degenerating photoreceptors,however, detached from the culture substratum). Cell numbers wereexpressed either as cells/6-mm well.

[0224] Cultured rat retinal cells enriched for photoreceptors(10,000/6-mm well) were treated with human recombinant GDNF (ten-foldserial dilutions ranging from 10 ng/mL to 1 pg/mL). The cultures werefixed after six days and immunostained for arrestin, a rodphotoreceptor-specific antigen. In cultures that were not treated withGDNF, the number of photoreceptors declined steadily over time to reachabout 25 percent of the initial number after six days in culture.Treatment of the cultures with GDNF resulted in an about two-fold highernumber of viable arrestin-positive photoreceptors after six days inculture. The effect of GDNF was maximal at about 200 pg/mL, with an ED₅₀of about 30 pg/mL. In addition to promoting photoreceptor survival, theaddition of the GDNF also stimulated the extension of their axon-likeprocess, thereby demonstrating an effect on the morphologicaldevelopment of the photoreceptors (mean neurite length of photoreceptorsin GDNF: 68 μm, compared to 27±18 μm in control cultures).

[0225] In order to confirm that rat retinal cells express high affinityGDNF receptors, [¹²⁵I]GDNF binding and photographic emulsion analysiswere carried out. Post-natal rat photoreceptor cells were seeded onplastic slide flaskettes (Nunc) at a density of 2800 cells/mm2, three tofour days before the experiments. The cells were washed once withice-cold washing buffer (Dulbecco's Modified Eagle's Medium (DMEM)containing 25 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid(HEPES), pH 7.5). For competitive binding, the cells were incubated withvarious concentrations of [¹²⁵I]GDNF in binding buffer (DMEM containing25 mM HEPES, pH 7.5, and 2 mg/mL of bovine serum albumin (BSA)) in thepresence or absence of 500 nM unlabeled GDNF at 4° C. for four hours.Cells were washed four times with ice-cold washing buffer, lysed in 1 MNaOH and the radioactivity associated with the cells was determined in agamma counter. A significant amount of [¹²⁵I]GDNF bound to thephotoreceptor cells even at low ligand concentrations (as low as 30 pM),and this binding was inhibited completely by the presence of excessunlabeled GDNF.

[0226] For photographic emulsion detection, cells were incubated with 50pM of [¹²⁵I]GDNF in binding buffer in the presence or absence of 500 nMunlabeled GDNF at 4° C. for four hours. Cells were washed six times withice-cold washing buffer, fixed with 2.5% glutaraldehyde and dehydratedsequentially with 50% and 70% ethanol, and dipped in NTB-2 photographicemulsion (Eastman Kodak, Rochester N.Y.). After five days of exposure,the slides were developed and examined. The photographic emulsionanalysis demonstrated the association of [¹²⁵I]GDNF to some of thephotoreceptor cells, thereby indicating the presence of a receptor forGDNF. This association, however, was efficiently blocked by the additionof unlabeled GDNF.

Example 2 Expression Cloning of a GDNFR from Photoreceptor Cells

[0227] Rat photoreceptor cells were selected as a possible source of ahigh affinity receptor for GDNF based upon their cell surface binding ofradiolabeled GDNF and their ability to respond to very lowconcentrations of the ligand, as described in Example 1. In order toidentify the receptor, a size-selected cDNA library of approximately50,000 independent clones was constructed using a mammalian expressionvector (a derivative of pSR, Takebe et al., 1988 supra) and mRNAisolated from cultured post-natal rat photoreceptor cells, by themethods described below. The library was divided into pools ofapproximately 1,500 to 2,000 independent clones and screened using anestablished expression cloning approach (Gearing et al., EMBO Journal,8, 3667-3676, 1989). Plasmid DNA representing each pool of the librarywas prepared and transfected into COS7 cells grown on plastic microscopeslide flaskettes (Nunc, Naperville, Ill.).

[0228] The transfected cells were treated with [¹²⁵I]GDNF, fixed withglutaraldehyde, dehydrated, and dipped in photographic emulsion forautoradiography. Following exposure for five days, the slides weredeveloped and examined for the presence of cells covered by silvergrains which indicated the binding of [¹²⁵I]GDNF to the cell surface asa result of the cell's expression of a receptor for GDNF. EGF receptortransfected cells treated with [¹²⁵I]EGF were used as a positivecontrol.

[0229] One of the 27 pools (F8-11) screened in this manner exhibited 19positive cells following transfection. Thus, a single cDNA library poolwas identified which contained a cDNA clone that expressed GDNFR. Thispool was divided into 60 smaller subpools of 100 clones/pool which wererescreened by the same procedure described above. Five of these poolswere identified as positive and two of the five pools were furthersubdivided to yield single clones responsible for the GDNF bindingactivity. Transfection of plasmid DNA from the single clones into COS7cells resulted in the binding of [¹²⁵I]GDNF to approximately 15% of thecells. This binding was specifically inhibited by competition withexcess unlabeled GDNF.

[0230] Construction of Expression cDNA Libraries

[0231] Rat retinal cells were harvested from postnatal day 3-7 rats andseeded into culture dishes coated with laminin and polyornithine at adensity of approximately 5700 cells/mm². After 3-4 days in culture, thepopulation was estimated to contain approximately 80% photoreceptorcells. Total RNA was prepared from this culture by standard methods, andPoly A+ RNA was purified using a polyA-tract kit (Promega, Madison,Wis.). A cDNA library was constructed from the rat photoreceptor poly A+RNA using the Gibco Superscript Choice System (Gibco/BRL, Gaithersburg,Md.). Two micrograms of poly A+ RNA were mixed with 50 ng of randomhexamers, heated to 70° C. for 10 minutes and then quick-chilled on ice.First strand synthesis was carried out with 400U Superscript II RT at37° C. for one hour. Second strand synthesis was performed in the sametube after the addition of dNTPs, 10U of E. coli DNA ligase, 40U of E.coli DNA polymerase I, and 2U of E. coli RNase H. After two hours at 16°C., the cDNA ends were blunted by treatment with 10U of T4 polymerasefor an additional five minutes at 16° C. Following isopropanolprecipitation, EcoRI cloning sites were added to the cDNA by ligationovernight with 10 μg of unphosphorylated EcoRI adapter oligonucleotides.

[0232] The EcoRI adapted cDNA was then phosphorylated and applied to aSephacryl S-500 HR size fractionation column. Following loading, thecolumn was washed with 100 μl aliquots of TEN buffer (10 mM Tris-HCl pH7.5, 0.1 mM EDTA, 25 mM NaCl), and 30 μl fractions were collected.Fractions 6 through 8, which contained approximately 34 ng of highmolecular weight cDNA, were pooled and precipitated. The recoveredEcoRI-adapted cDNA was ligated overnight with 50 ng of EcoRI cut vectorpBJ5. Aliquots of the ligation mix containing about 15 ng cDNA each weretransformed into competent cells (E. coli strain DH10B; GIBCO/BRL,Gaithersburg, Md.) by electroporation. The transformation mixture wastitered and then plated on 27 Amp/LB plates at a density of 1500colonies/plate. Colonies were scraped from each plate and collected into10 mL of Luria broth (LB) to make 27 pools of 1500 independent cloneseach. A portion of the cells from each pool was frozen in glycerol andthe remainder was used to isolate plasmid DNA using a Qiagen tip-500 kit(Qiagen Inc., Chatsworth, Calif.).

[0233] COS Cell Transfection and Photographic Emulsion Analysis

[0234] COS7 cells were seeded (220,000 cells/slide) on plastic slideflaskettes (Nunc) coated with ProNectin (10 μg/mL in phosphate bufferedsaline (PBS)) one day before transfection. For transfection, 700 μl ofOpti MEMI (GIBCO/BRL, Gaithersburg, Md.) containing 2 μg cDNA was mixedgently with 35 μl of DEAE Dextran solution (10 mg/mL, Sigma, St. Louis,Mo.) in an Eppendorf tube. Cells were washed twice with PBS andincubated with the transfection mix for 30 minutes at 37° C. in a 5% CO₂atmosphere. Following incubation, 3 mL of DMEM media containing 10%fetal calf serum (FCS) and 80 nM Chloroquine (Sigma, St. Louis, Mo.)were added to each flaskette. Cells were further incubated for 3.5hours, shocked with 10% dimethylsulfoxide in DMEM at room temperaturefor two minutes, washed once with PBS, and allowed to grow in DMEMcontaining 10% FCS. After 48 hours, the transfected COS7 cells werewashed once with ice-cold washing buffer (DMEM containing 25 mM HEPES,pH 7.5) and incubated in ice-cold binding buffer (DMEM containing 25 mMHEPES, pH 7.5 and 2 mg/mL BSA) supplemented with 50 pM [¹²⁵I]GDNF at 4°C. for four hours. Cells were washed six times in ice-cold washingbuffer, fixed with 2.5% glutaraldehyde at room temperature for fiveminutes, dehydrated sequentially with 50% and 70% ethanol, and thendipped in NTB-2 photographic emulsion (Eastman Kodak). After 4-5 dayexposure at 4° C. in dark, the slides were developed and screened bybright-field and dark-field microscopy.

[0235] Subdivision of Positive Pools

[0236] A single pool was identified which contained a putative GDNFreceptor clone. Clones from this pool were plated on 60 plates at adensity of 100 colonies/plate. Cells were scraped from each plate,collected in LB, and allowed to grow for 4-5 hours at 37° C. Frozenstocks and DNA preparations were made from each pool, as before, togenerate 60 subpools containing 100 independent clones each. Two ofthese 60 subpools were identified as positive by the method describedabove, and clones from those pools were plated at low density to allowisolation of single colonies. Single colonies (384) were picked fromeach of the two subpools and grown for six hours in 200 μl LB in 96-wellplates. In order to select single clones expressing GDNFR, the four96-well plates were arrayed into a single large matrix consisting of 16rows and 24 columns. Cells from the wells in each row and in each columnwere combined to yield a total of 40 mixtures. These mixtures were grownovernight in 10 mL LB/Amp (100 μg/mL), and DNA was prepared using aQiagen tip-20 kit. When analyzed for putative GDNF receptor clones,three row mixtures and three column mixtures gave positive signals,suggesting nine potentially positive single clones. DNA from each of thepotentially positive single clones was prepared and digested with EcoRIand PstI. DNA from three of the nine single clones exhibited identicalrestriction patterns while the other six were unrelated, suggesting thatthe three represented the authentic clones containing GDNFR.

Example 3 DNA Sequencing and Sequence Analysis

[0237] DNA from positive, single clones was prepared and sequenced usingan automated ABI373A DNA sequencer (Perkin/Elmer Applied Biosystems,Santa Clara, Calif. ) and dideoxy-dye-terminators, according tomanufacturer's instructions. Comparison of GDNF receptor sequence withall available public databases was performed using the FASTA (Pearsonand Lipman, Proceedings Of The National Academy Of Sciences U.S.A., 85,2444-2448, 1988) program algorithm as described in the University ofWisconsin Genetics Computer Group package (Program Manual for theWisconsin Package, Version 8, September 1994, Genetics Computer Group,Madison, Wis.).

[0238] Sequence Characterization of the Rat GDNFR

[0239] Plasmid DNA from the clones described in Example 2, above, wasprepared and submitted for DNA sequence analysis. Nucleotide sequenceanalysis of the cloned 2138 bp rat cDNA revealed a single large openreading frame encoding a translation protein of 468 amino acid residues(FIG. 3).

[0240] The coding sequence is flanked by a 5′-untranslated region of 301bp and a 3′-untranslated region of 430 bp that does not contain apotential polyadenylation site. The presence of an in-frame stop codonupstream of the first ATG at base pair 302 and its surroundingnucleotide context indicate that this methionine codon is the mostlikely translation initiator site (Kozak, Nucleic Acids Research. 15,8125-8148, 1987).

[0241] No polyadenylation signal is found in the 430 nucleotides of 3′untranslated sequence in the rat cDNA clone. This is not surprising,since the Northern blot data shows the shortest mRNA transcripts to beapproximately 3.6 kb.

[0242] The GDNFR polypeptide sequence has an N-terminal hydrophobicregion of approximately 19 residues (methionine-1 to alanine-19, FIG. 3)with the characteristics of a secretory signal peptide (von Heijne,Protein Sequences And Data Analysis. 1, 41-42, 1987; von Heijne, NucleicAcids Research. 14, 4683-4690, 1986). No internal hydrophobic domainthat could serve as a transmembrane domain was found. Instead, acarboxy-terminal hydrophobic region of 21 residues (leucine-448 toserine-468 in FIG. 3) is present and may be involved in aglycosyl-phosphatidylinositol (GPI) anchorage of the receptor to thecytoplasmic membrane. Except for the presence of three potentialN-linked glycosylation sites, no conserved sequence or structural motifswere found. The protein is extremely rich in cysteine (31 of the 468amino acid residues) but their spacing is not shared with that ofcysteine-rich domains found in the extracellular portions of knownreceptors.

[0243] The GDNFR sequence was compared to sequences in available publicdatabases using FASTA. The search did not reveal significant homology toother published sequences. Once the rat cDNA clone was obtained, it wasradiolabeled and used to probe a cDNA library prepared from human brainsubstantia nigra as described below in Example 5.

Example 4 GDNF Binding to Cells Expressing GDNFR

[0244] A binding assay was performed in accordance with an assay methodpreviously described by Jing et al. (Journal Of Cell Biology, 110,283-294, 1990). The assay involved the binding of [¹²⁵I]GDNF to ratphotoreceptor cells, COS7 cells or 293T cells which had been transfectedto express GDNFR. Recombinant GDNFR expressed on the surface of 293Tcells was able to bind GDNF specifically and with an affinity comparableto that observed for GDNF binding sites on rat retinal cells.

[0245] Rat photoreceptor cells were prepared as described in Example 1,above, and seeded at a density of 5.7×10⁵ cells/cm² two to three daysbefore the assay in 24-well Costar tissue culture plates pre-coated withpolyornithine and laminin. COS7 cells were seeded at a density of2.5×10⁴ cells/cm² one day before the assay and transfected with 10-20 μgof plasmid DNA using the DEAE-dextran-chloroquine method (Aruffo andSeed, Proceedings Of The National Academy Of Sciences U.S.A., 84,8573-8577, 1987). Cells from each dish were removed and reseeded into 30wells of 24-well Costar tissue culture plates 24 hours following thetransfection, and allowed to grow for an additional 48 hours. Cells werethen left on ice for 5 to 10 minutes, washed once with ice-cold washingbuffer and incubated with 0.2 mL of binding buffer containing variousconcentrations of [¹²⁵I]GDNF with or without unlabeled GDNF at 4° C. forfour hours. Cells were washed four times with 0.5 mL ice-cold washingbuffer and lysed with 0.5 mL of 1 M NaOH. The lysates were counted in a1470 Wizard Automatic Gamma Counter.

[0246] For some binding experiments, transiently transfected 293T cellswere used (see below for 293T cell transfection). Two days followingtransfection, cells were removed from dishes by 2× versine. Cells werepelleted, washed once with ice-cold binding buffer and resuspended inice-cold binding buffer at a density of 3×10⁵ cells/mL. The cellsuspension was divided into aliquots containing 1.5×10⁵ cell/sample.Cells were then pelleted and incubated with various concentrations of[¹²⁵I]GDNF in the presence or absence of 500 nM of unlabeled GDNF at 4°C. for four hours with gentle agitation. Cells were washed four timeswith ice-cold washing buffer and resuspended in 0.5 mL washing buffer.Two 0.2 mL aliquots of the suspension were counted in a gamma counter todetermine the amount of [¹²⁵I]GDNF associated with the cells.

[0247] In all assays, nonspecific binding was determined by usingduplicate samples, one of which contained 500 nM of unlabeled GDNF. Thelevel of nonspecific binding varied from 10% to 20% of the specificbinding measured in the absence of unlabeled GDNF and was subtractedfrom the specific binding. The assays demonstrated that cells did notbind GDNF unless the cell had been transfected with the GDNFR cDNAclone.

Example 5 Tissue Distribution of GDNFR mRNA

[0248] The pattern of expression of GDNFR mRNA in embryonic mouse, adultmouse, rat, and human tissues was examined by Northern blot analysis.The cloned rat GDNFR cDNA was labeled using the Random Primed DNALabeling Kit (Boehringer Mannheim, Indianapolis, Ind.) according to themanufacturer's procedures. Rat, mouse, and human tissue RNA blots(purchased from Clontech, Palo Alto, Calif.) were hybridized with theprobe and washed using the reagents of the ExpressHyb Kit (Clontech)according to the manufacturer's instructions.

[0249] Tissue Northern blots prepared from adult rat, mouse, and humantissues indicated that GDNFR mRNA is most highly expressed in liver,brain, and kidney. High mRNA expression was also detected in lung, withlower or non-detectable amounts in spleen, intestine, testis, andskeletal muscle. In blots made from mRNA isolated from mouse embryo,expression was undetectable at embryonic day 7, became apparent at dayE11, and was very high by day E17. GDNFR mRNA was expressed in tissueisolated from several subregions of adult human brain at relativelyequal levels. Expression of GDNFR mRNA in human adult brain showedlittle specificity for any particular region.

[0250] In most tissues, transcripts of two distinct sizes were present.In mouse and human tissues, transcripts of 8.5 and 4.4 kb were found,while in rat the transcripts were 8.5 and 3.6 kb. The relative amountsof the larger and smaller transcripts varied with tissue type, thesmaller transcript being predominant in liver and kidney and the largerbeing more abundant in brain. The binding of GDNF to 293T cellstransfected with a GDNFR cDNA clone in the pBKRSV vector was examined byScatchard analysis. Two classes of binding sites were detected, one witha binding affinity in the low picomolar range and another with anaffinity of about 500 pM.

Example 6 Recombinant Human GDNFR

[0251] An adult human substantia nigra cDNA library (5′-stretch pluscDNA library, Clontech, Palo Alto, Calif.) cloned in bacteriophage gt10was screened using the rat GDNFR cDNA clone of Example 1 as a probe. Theprobe was labeled with [³²P]-dNTPs using a Random Primed DNA LabelingKit (Boehringer Mannheim, Indianapolis, Ind.) according to themanufacturer's instructions. Approximately 1.2×10⁶ gt10 phage from thehuman substantia nigra cDNA library were plated on 15 cm agarose platesand replicated on duplicate nitrocellulose filters. The filters werethen screened by hybridization with the radiolabeled probe. The filterswere prehybridized in 200 mL of 6× SSC, 1× Denhardts, 0.5% SDS, 50 μg/mLsalmon sperm DNA at 55° C. for 3.5 hours. Following the addition of2×10⁸ cpm of the radiolabeled probe, hybridization was continued for 18hours. Filters were then washed twice for 30 minutes each in 0.5× SSC,0.1% SDS at 55° C. and exposed to X-ray film overnight with anintensifying screen.

[0252] Five positive plaques were isolated whose cDNA insertsrepresented portions of the human GDNFR cDNA. In comparison to thenucleic acid sequence of rat GDNFR depicted in FIG. 3 (bp 0 through2140), the five human GDNFR clones were found to contain the followingsequences: TABLE 3 Clone 2 1247 through 2330 (SEQ ID NO:21) Clone 9 1270through 2330 (SEQ ID NO:23) Clone 21-A −235 through 1692 (SEQ ID NO:9)Clone 21-B −237 through 1692 (SEQ ID NO:l 1) Clone 29  805 through 2971(SEQ ID NO:15)

[0253] An alignment and comparison of the sequences, as depicted in FIG.5, provided a consensus sequence for human GDNFR. The translationproduct predicted by the human cDNA sequence consists of 465 amino acidsand is 93% identical to rat GDNFR.

[0254] To generate a human cDNA encoding the full length GDNFR, portionsof clones 21B and 2 were spliced together at an internal BglII site andsubcloned into the mammalian expression vector pBKRSV (Stratagene, LaJolla, Calif.).

[0255] Recombinant human GDNFR expression vectors may be prepared forexpression in mammalian cells. As indicated above, expression may alsobe in non-mammalian cells, such as bacterial cells. The nucleic acidsequences disclosed herein may be placed into a commercially availablemammalian vector (for example, CEP4, Invitrogen) for expression inmammalian cells, including the commercially available human embryonickidney cell line, “293”. For expression in bacterial cells, one wouldtypically eliminate that portion encoding the leader sequence (e.g.,nucleic acids 1-590 of FIG. 1). One may add an additional methionyl atthe N-terminus for bacterial expression. Additionally, one maysubstitute the native leader sequence with a different leader sequence,or other sequence for cleavage for ease of expression.

Example 7 Soluble GDNFR Constructs

[0256] Soluble human GDNFR protein products were made. The followingexamples provide four different forms, differing only at the carboxyterminus, indicated by residue numbering as provided in FIG. 2. Two aresoluble forms truncated at different points just upstream from thehydrophobic tail and downstream from the last cysteine residue. Theother two are the same truncations but with the addition of the “FLAG”sequence, an octapeptide to which a commercial antibody is available(Eastman Kodak). The FLAG sequence is H2N-DYKDDDDK-COOH.

[0257] Method

[0258] Lambda phage clone #21, containing nearly the entire codingregion of human GDNFR, was digested with EcoRI to excise the cDNAinsert. This fragment was purified and ligated into EcoRI cut pBKRSVvector (Stratagene, La Jolla, Calif.) to produce the clone21-B-3/pBKRSV. Primers 1 and 2 as shown below were used in a PCRreaction with the human GDNFR clone 21-B-3/pBKRSV as template. PCRconditions were 94° C., five minutes followed by 25 cycles of 94° C.,one minute; 55° C., one minute; 72° C., two minutes and a finalextension of five minutes at 72° C. This produced a fragment consistingof nucleotides 1265-1868 of the human GDNFR clone plus a terminationcodon and Hind III restriction site provided by primer 2. This fragmentwas digested with restriction enzymes Hind III (contained in primer 2)and BglII (position 1304 in human GDNFR), and the resulting 572nucleotide fragment was isolated by gel electrophoresis. This fragmentcontained the hGDNFR-coding region from isoleucine-255 to glycine-443. Asimilar strategy was used with primers 1 and 3 to produce a fragmentwith BglII and HindIII ends which coded for isoleucine-255 toproline-446. Primers 4 and 5 were designed to produce fragments codingfor the same regions of hGDNFR and primers 1 and 3, but with theaddition of the Flag peptide coding sequence (IBI/Kodak, New Haven,Conn.). The Flag peptide sequence consists of eight amino acids(H2N-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-COOH) to which antibodies arecommercially available. Primers 1 and 4 or 1 and 5 were used in PCRreactions with the same template as before, and digested with HindIIIand BglII as before. This procedure produced fragments coding forisoleucine-255 to glycine-443 and isoleucine-255 to proline-446, butwith the addition of the Flag peptide at their carboxy termini.Primers 1) 5′-CTGTTTGAATTTGCAGGACTC-3′ (SEQ ID NO:30) 2)5′-CTCCTCTCTAAGCTTCTAACCACAGCTTGGAGGAGC3′ (SEQ ID NO:31) 3)5′-CTCCTCTCTAAGCTTCTATGGGCTCAGACCACAGCTT-3′ (SEQ ID NO:32) 4)5′-CTCCTCTCTAAGCTTCTACTTGTCATCGTCGTCCTTGTAGTCACCACAGCTTGGAGGAGC-3′ (SEQID NO:33) 5)5′-CTCCTCTCTAAGCTTCTACTTGTCATCGTCGTCCTTGTAGTCTGGCTCAGACCACAGCTT-3′ (SEQID NO:34)

[0259] All four fragments, produced as described above, were transferredback into 21B3/pBKRSV. The 21B3/pBKRSV clone was digested with BglII andHindIII, and treated with calf intestinal alkaline phosphatase (CIAP).The large fragment containing the vector and the human GDNFR codingregion up to the BglII site was gel purified and extracted from gel.Each of the four BglII/HindIII fragments produced as described abovewere ligated into this vector resulting in the following constructs inthe pBKRSV vector: TABLE 4 1) GDNFR/gly-443/pBKRSV hGDNFR terminating atglycine 443, followed by stop codon 2) GDNFR/pro-446/pBKRSV hGDNFRterminating at proline 446, followed by stop codon 3) GDNFR/gly- hGDNFRterminating at glycine 443 443/Flag/pBKRSV with C-term Flag tag,followed by stop codon 4) GDNFR/pro- hGDNFR terminating at proline 446446/Flag/pBKRSV with C-term Flag tag, followed by stop codon

[0260] Correct construction of all clones was confirmed by DNAsequencing. Inserts from the pBKRSV clones were transferred to otherexpression vectors using enzyme sites present in the pBKRSV polylinkersequence as described below. Soluble GDNFRs (e.g., sGDNFR/gly andsGDNFR/pro) have also been transferred into vectors for transientexpression and into pDSR-2 for stable expression in CHO cells.

[0261] pDSRα2+PL Clones:

[0262] The appropriate pBKRSV clone is digested with XbaI and SalI. Theinsert is ligated to pDSRα2+PL cut with the same enzymes and treatedwith CIAP. This construction may be used for stable expression of GDNFRin CHO cells.

[0263] pCEP4 Clones:

[0264] The appropriate pBKRSV clone is digested with SpeI and XhoI. Theinsert is ligated to pCEP4 (Invitrogen, San Diego, Calif.) digested withNheI (SpeI ends) and XhoI, and treated with CIAP. This construction maybe used for transient of expression of GDNFR.

[0265] The plasmid construct pDSR-2 is prepared substantially inaccordance with the process described in the co-owned and copending U.S.patent application Ser. No. 501,904 filed Mar. 29, 1990 (also see,European Patent Application No. 90305433, Publication No. EP 398 753,filed May 18, 1990 and WO 90/14363 (1990), the disclosures of which arehereby incorporated by reference. It will be appreciated by thoseskilled in the art that a variety of nucleic acid sequences encodingGDNFR analogs may be used.

[0266] Another construct is pDSRα2, a derivative of the plasmid pCD(Okayama & Berg, Mol. Cell Biol. 3: 280-289, 1983) with three mainmodifications: (i) the SV40 polyadenylation signal has been replacedwith the signal from the α-subunit of bovine follicular stimulatinghormone, α-bFSH (Goodwin et al., Nucleic Acids Res. 11: 6873-6882,1983); (ii) a mouse dihydrofolate reductase minigene (Gasser et al.,Proc. Natl. Acad. Sci. 79: 6522-6526, 1982) has been inserted downstreamfrom the expression cassette to allow selection and amplification of thetransformants; and (iii) a 267 bp fragment containing the “R-element”and part of the “U5” sequences of the long terminal repeat (LTR) ofhuman T-cell leukemia virus type I (HTLV-I) has been cloned and insertedbetween the SV40 promoter and the splice signals as described previously(Takebe et al., Mol. Cell Biol. 8: 466-472, 1988).

[0267] The expression of GDNFR in CHO cells has been verified by thebinding of iodinated GDNF to the cell surface. As discussed above, therecombinantly expressed soluble GDNFR protein product may be used topotentiate the activity or cell specificity of GDNF. Soluble GDNFRattached to a detectable label also may be used in diagnosticapplications as discussed above.

Example 8 Chemical Crosslinking of GDNF with GDNFR

[0268] In order to study its binding properties and molecularcharacteristics, GDNFR was transiently expressed on the surface of 293Tcells by transfection of the rat cDNA clone. Transfection of 293T cellswas performed using the Calcium Phosphate Transfection System(GIBCO/BRL, Gaithersburg, Md.) according to the manufacturersinstructions. Two days following transfection, cells were removed by 2×versine treatment, washed once with washing buffer, and resuspended inwashing buffer at a density of 2×10⁶ cells/mL. A duplicate set of cellswere incubated with 0.5 u/mL PI-PLC at 37° C. for 30 minutes before[¹²⁵I]GDNF binding. These cells were washed three times with ice-coldbinding buffer and then incubated with 1 to 3 nM of [¹²⁵I]GDNF alongwith other cells at 4° C. for four hours. Cells were washed four timeswith ice-cold washing buffer, resuspended in washing buffer supplementedwith 1 mM of Bis suberate for crosslinking (BS³ Pierce, Rockford, Ill.)and incubated at room temperature for 30 minutes. Following three washeswith TBS, a duplicate group of samples was treated by 0.5 u/mL of PI-PLCat 37° C. for 30 minutes. These cells were pelleted and the supernatantswere collected. The cells were then washed with washing buffer and lysedalong with all other cells with 2× SDS-PAGE sample buffer. The celllysates and the collected supernatants were resolved on a 7.5% SDS-PAGE.

[0269] The cell suspension was divided into aliquots containing 1.5×10⁵cell/sample. Cells were then pelleted and incubated with variousconcentrations of [¹²⁵I]GDNF in the presence or absence of 500 nM ofunlabeled GDNF at 4° C. for four hours with gentle agitation. Cells werewashed four times with ice-cold washing buffer and resuspended in 0.5 mLwashing buffer. Two 0.2 mL aliquots of the suspension were counted in agamma counter to determine the amount of [¹²⁵I]GDNF associated with thecells.

[0270] Although mock transfected 293T cells did not exhibit any GDNFbinding capacity, GDNFR transfected cells bound [¹²⁵I]GDNF strongly evenat picomolar concentrations. This binding was almost completelyinhibited by 500 nM of unlabeled GDNF, indicating a specific binding ofnative GDNF to the expressed receptors.

[0271] GDNFR expressed by the 293T cells can be released from the cellsby treatment with phosphatidylinositol-specific phospholipase C (PI-PLC,Boehringer Mannheim, Indianapolis, Ind.). The treatment of transfectedcells with PI-PLC prior to ligand binding almost entirely eliminated theGDNF binding capacity of the cell. Additionally, treatment of thetransfected cells after cross-linking released the majority of thecross-linked products into the media. These results strongly suggestthat GDNFR is anchored to the cell membrane through a GPI linkage.

[0272] Crosslinking data further indicated that the molecular weight ofGDNFR is approximately 50-65 kD, suggesting that there is a low level ofglycosylation. Although the major cross-linked species has a molecularmass consistent with a monomer of the receptor, a minor species withapproximately the mass expected for a dimer has been found.

Example 9 GDNF Signaling is Mediated by a Complex of GDNFR and the RetReceptor Protein Tyrosine Kinase Introduction

[0273] Mice carrying targeted null mutations in the GDNF gene exhibitvarious defects in tissues derived from neural crest cells, in theautonomic nervous system, and in trigeminal and spinal cord motorneurons. The most severe defects are the absence of kidneys and completeloss of enteric neurons in digestive tract. The phenotype of GDNFknockout mice is strikingly similar to that of the c-ret knockoutanimals (Schuchardt et al. 1994), suggesting a possible linkage betweenthe signal transduction pathways of GDNF and c-ret.

[0274] The proto-oncogene c-ret was identified using probes derived froman oncogene isolated in gene transfer experiments (Takahashi et al.,Cell. 42, 581-588, 1985; Takahashi and Cooper, Mol. Cell. Biol., 7,1378-1385, 1987). Sequence analysis of the c-ret cDNA revealed a largeopen reading frame encoding a novel receptor protein tyrosine kinase(PTK). The family of receptor PTKs has been grouped into sub-familiesaccording to extracellular domain structure and sequence homology withinthe intracellular kinase domain (van der Geer et al., 1994). The uniqueextracellular domain structure of Ret places it outside any other knownreceptor PTK sub-family; it includes a signal peptide, a cadherin-likemotif, and a cysteine-rich region (van Heyningen, Nature, 367, 319-320,1994; Iwamoto et al., 1993). In situ hybridization andimmunohistochemical analysis showed high level expression of ret mRNAand protein in the developing central and peripheral nervous systems andin the excretory system of the mouse embryo (Pachnis et al., 1993;Tsuzuki et al., Oncogene, 10, 191-198, 1995), suggesting a role of theRet receptor either in the development or in the function of thesetissues. A functional ligand of the Ret receptor has not beenidentified, thereby limiting a further understanding of the molecularmechanism of Ret signaling. Mutations in the c-ret gene are associatedwith inherited predisposition to cancer in familial medullary thyroidcarcinoma (FMTC), and multiple endocrine neoplasia type 2A (MEN2A) and2B (MEN2B). These diseases are probably caused by “gain of function”mutations that constitutively activate the Ret kinase (Donis-Keller etal., Hum. Molec. Genet. 2, 851-856, 1993; Hofstra et al., Nature. 367,375-376, 1994; Mulligan et al., Nature. 363, 458-460, 1993; Santoro etal., Science. 267, 381-383, 1995). They confer a predisposition tomalignancy specifically in tissues derived from the neural crest, whereret is normally expressed in early development. Another ret-associatedgenetic disorder, Hirschsprung's disease (HSCR), is characterized by thecongenital absence of parasympathetic innervation in the lowerintestinal tract (Edery et al., Nature. 367, 378-380, 1994; Romeo etal., 1994). The most likely causes of HSCR are nonsense mutations thatresult in the production of truncated Ret protein lacking a kinasedomain or missense mutations that inactivate the Ret kinase. As notedabove, targeted disruption of the c-ret proto-oncogene in mice resultsin renal agenesis or severe dysgenesis and lack of enteric neuronsthroughout the digestive tract (Schuchardt et al., 1994). This phenotypeclosely resembles that of GDNF knockout mice. Taken together, these datasuggest that both Ret and GDNF are involved in signal transductionpathways critical to the development of the kidney and the entericnervous system. How Ret and GDNF are involved, however, was not known.

[0275] The isolation and characterization of cDNA for GDNFR byexpression cloning, as described above, lead to the expression of GDNFRin the transformed human embryonic kidney cell line 293T. Transformationresulted in the appearance of both high (K_(d) of approximately 2 pM)and low (K_(d) of approximately 200 pM) affinity binding sites. The highaffinity binding sites could be composed of homodimers or homo-oligomersof GDNFR alone, or of heterodimers or hetero-oligomers of GDNFR withother molecules. As discussed above, because GDNFR lacks a cytoplasmicdomain, it must function through one or more accessory molecules inorder to play a role in GDNF signal transduction. In this study weconfirm that, in the presence of GDNFR, GDNF associates with the Retprotein tyrosine kinase receptor, and quickly induces Retautophosphorylation.

Results

[0276] Neuro-2a Cells Expressing GDNFR Bind GDNF with High Affinity

[0277] Neuro-2a is a mouse neuroblastoma cell line that endogenouslyexpresses a high level of Ret protein (Ikeda et al., Oncogene. 5,1291-1296, 1990; Iwamoto et al., Oncogene. 8, 1087-1091, 1993; Takahashiand Cooper, 1987) but does not express detectable levels of GDNFR mRNAas judged by Northern blot. In order to determine if Ret could associatewith GDNF in the presence of GDNFR, a study was performed to examine thebinding of [¹²⁵I]GDNF to Neuro-2a cells engineered to express GDNFR.Neuro-2a cells were transfected with a mammalian expression vectorcontaining the rat GDNFR cDNA (such as the expression plasmid describedabove). Three clonal lines, NGR-16, NGR-33, and NGR-38 were tested fortheir ability to bind [¹²⁵I]GDNF. The unbound [¹²⁵I]GDNF was removed atthe end of the incubation and the amount of radioactivity associatedwith the cells was determined as described in Experimental Procedures.All three lines were able to bind [¹²⁵I]GDNF specifically while parentalNeuro-2a cells exhibited little or no [¹²⁵I]GDNF binding (FIG. 6).Binding could be effectively competed by the addition of 500 nMunlabeled GDNF. These results demonstrate that Ret receptor expressed onNeuro-2a cells is unable to bind GDNF in the absence of GDNFR and areconsistent with the previous observation that GDNFR is not expressed atappreciable levels in Neuro-2a cells.

[0278] Equilibrium binding of [¹²⁵I]GDNF to NGR-38 cells was examinedover a wide range of ligand concentrations (0.5 pM to 1 nM of [¹²⁵I]GDNFin the presence or absence of 500 nM of unlabeled GDNF) (see FIG. 7A).Following incubation, unbound [¹²⁵I]GDNF was removed and theradioactivity associated with the cells was determined as described inExperimental Procedures. Results are depicted in FIG. 7: (A) Equilibriumbinding of [¹²⁵I]GDNF to NGR-38 cells (circles) and Neuro-2a cells(squares) in the presence (open circles and open squares) or absence(filled circles and filled squares) of unlabeled GDNF; (B) Scatchardanalysis of [¹²⁵I]GDNF binding to NGR-38 cells. Neuro-2a cells exhibitedlittle binding even at a concentration of 1 nM [¹²⁵I]GDNF, and thisbinding was not affected by the addition of excess unlabeled GDNF.Binding to NGR-38 cells was analyzed by Scatchard plot as shown in FIG.7B. Two classes of binding sites were detected, one with K_(d)=1.5 ±0.5pM and the other with K_(d)=332 ±53 pM. These dissociation constants arevery similar to the values obtained for the high and low affinitybinding sites in 293T cells transiently expressing GDNFR, as describedabove.

[0279] GDNF Associates with Ret in Neuro-2a Cells Expressing GDNFR

[0280] In order to determine if the Ret receptor PTK could associatewith GDNF in cells expressing GDNFR, a cross-linking experiment wascarried out using NGR-38 and parental Neuro-2a cells. NGR-38 cells wereincubated with [¹²⁵I]GDNF, treated with cross-linking reagent, thenlysed either directly in SDS-PAGE sample buffer or in Triton X-100 lysisbuffer and further immunoprecipitated with anti-Ret antibody asdescribed in the Experimental Procedures. The immunoprecipitates wereanalyzed by SDS-PAGE in the absence (NR) or presence (R) of-mercaptoethanol. Lysates were treated with Ret specific antibody,immunoprecipitated, and analyzed by SDS-PAGE under reducing conditions(see FIG. 8, bands are marked as follows: ˜75 kD, solid triangle; ˜150kD, open triangle; ˜185 kD, solid arrow; ˜250 kD, asterisk; ˜400kD, openarrow). The most prominent cross-linked species were at ˜75 kD, and ˜185kD, with less intense bands of ˜150 kD and ˜250 kD. A very faint band of˜400 kD was also visible (FIG. 8, lane 2). When immunoprecipitates wereanalyzed by non-reducing SDS-PAGE, the ˜75 kD, ˜150 and ˜185 kD bandswere present at about the same intensity as in the reducing gel, but theamount of the ˜400 kD band increased dramatically (FIG. 8, lane 4). Alsobecoming more prominent was the band at ˜250 kD.

[0281] Under both reducing and non-reducing conditions, bands of similarmolecular weight but of greatly reduced intensity were observed whenparental Neuro-2a cells were used instead of NGR-38 (FIG. 8, lanes 1 and3). The ˜75 kD and ˜150 kD species are likely to represent cross-linkedcomplexes of GDNF and GDNFR, since species with identical molecularweights are produced by cross-linking in 293T cells that do not expressRet. Furthermore, since the molecular weight of Ret is 170 kD, anycomplex including Ret must be of at least this size.

[0282] The fact that these complexes are immunoprecipitated by anti-Retantibody indicates they are products of an association between Ret andthe GDNF/GDNFR complex which was disrupted under the conditions of thegel analysis. It is envisioned that the broad band at ˜185 kD probablyconsists of one molecule of Ret (170 kD) cross-linked with one moleculeof monomeric recombinant GDNF (15 kD), although some dimeric GDNF may beincluded. The presence of Ret in this species was confirmed by aseparate experiment in which a band of the same molecular weight wasobserved when unlabeled GDNF was cross-linked to NGR-38 cells and theproducts examined by Western blot with anti-Ret antibody (data notshown).

[0283] The ˜400 kD band was not reliably identified, partly due to thedifficulty in estimating its molecular weight. The fact that it isprominent only under non-reducing conditions indicates that it is adisulfide-linked dimer of one or more of the species observed underreducing conditions. The most likely explanation is that it represents adimer of the 185 kD species, although it may be a mixture of highmolecular weight complexes consisting of two Ret, one or two GDNFR, andone or two GDNF molecules. The exact identity of the ˜250 kD band hasnot yet been determined. One possibility is that it representscross-linked heterodimers of the ˜75 kD (GDNF+GDNFR) and ˜185 kD(GDNF+Ret) complexes.

[0284] GDNF Stimulates Autophosphorylation of Ret in Neuro-2a CellsExpressing GDNFR

[0285] The ability of the Ret protein tyrosine kinase receptor toassociate with GDNF in the presence of GDNFR led to the study of GDNFstimulation of the autophosphorylation of Ret. NGR-38 cells were treatedwith GDNF, lysed, and the lysates immunoprecipitated with anti-Retantibody. The immunoprecipitates were analyzed by Western blot using ananti-phosphotyrosine antibody as described in the ExperimentalProcedures. When NGR-38 cells (FIG. 9A, lanes 2-4) were treated withpurified recombinant GDNF produced in either mammalian (CHO cells; FIG.9A, lanes 4) or E. coli cells (FIG. 9A, lanes 1, 3), a strong band wasobserved at 170 kD, indicating autophosphorylation of tyrosine residueson the mature form of Ret. A much weaker corresponding band was observedin GDNF-treated Neuro-2a cells (FIG. 9A, lane 1). No phosphorylation wasobserved on the alternatively glycosylated 150 kD precursor form of Ret(FIG. 9A). The induction of Ret autophosphorylation by GDNF was dosagedependent. The dose response and kinetics of GDNF-induced Ret tyrosinephosphorylation in NGR-38 cells are shown in panels B and C. In allpanels, the tyrosine phosphorylated 170 kD Ret bands are indicated bysolid arrows. The amount of Ret protein loaded in each lane asdetermined by reprobing of the immunoblot with anti-Ret antibody (SantaCruz, C-19, Cat. #sc-167) is shown on the right side of panel A. Theband at ˜150 kD represents an alternately glycosylated immature form ofRet that does not autophosphorylate. As shown in FIG. 9B, stimulation ofRet autophosphorylation in NGR-38 cells could be detected with 50 pg/mLof GDNF and the response was saturated at 20-50 ng/mL GDNF. Thestimulation of Ret autophosphorylation by purified recombinant GDNF inNGR-38 cells over times of 0-20 minutes following treatment is shown inFIG. 9C. Increased levels of Ret autophosphorylation could be observedwithin one minute of GDNF treatment and was maximal at 10 minutesfollowing treatment (FIG. 9C).

[0286] GDNF and Soluble GDNFR Induce Ret Autophosphorylation in Neuro-2ACells

[0287] As discussed above, GDNFR is anchored to the cytoplasmic membranethrough a GPI linkage and can be released by treatment withphosphatidylinositol-specific phospholipase C (PI-PLC). When NGR-38cells were incubated with PI-PLC, GDNF-induced receptorautophosphorylation of Ret in these cells was abolished (FIG. 10A;PI-PLC treated (lane 1) or untreated (lanes 2 and 3) NGR-38 cells wereincubated with (lanes 1 and 3) or without (lane 2) GDNF and analyzed forRet autophosphorylation by immunoblotting as described in theExperimental Procedures).

[0288]FIG. 10B depicts parental Neuro-2a cells treated with (lanes2,4,6,8) or without (lanes 1,3,5,7) GDNF in the presence (lanes 5-8) orabsence (lanes 1-4) of PI-PLC/CM obtained from Neuro-2a or NGR-38 cells,as analyzed for Ret autophosphorylation by immunoblotting as describedin the Experimental Procedures. NGR-38 cells treated with GDNF were usedas a positive control. In both panels A and B, the autophosphorylated170 kD Ret bands are marked by solid arrows. When conditioned mediumcontaining soluble GDNFR released by PI-PLC treatment (PI-PLC/CM) ofNGR-38 cells was added to parental Neuro-2a cells along with GDNF,autophosphorylation of the Ret receptor comparable to that obtained withGDNF treatment of NGR-38 cells was observed (FIG. 10B, lanes 2 and 8).Only background levels of Ret autophosphorylation were observed when noGDNF was added, or when conditioned media derived from PI-PLC treatmentof Neuro-2a cells was tested (FIG. 10B, lanes 3-7).

[0289] Ret-Fc Fusion Protein Blocks Ret Phosphorylation Induced by GDNFand Soluble GDNFR

[0290] To confirm that Ret phosphorylation induced by GDNF in thepresence of GDNFR is the result of receptor autophosphorylation, a studywas performed to determine whether a Ret extracellulardomain/Immunoglobulin Fc (Ret-Fc) fusion protein could block Retactivation. Because of the technical difficulty of blocking the largenumber of GDNF alpha receptors expressed on NGR-38 cells, Retphosphorylation assays were performed using Neuro-2a as the target celland culture media removed from NGR-38 cells treated with PI-PLC as asource of GDNFR. Cells were treated with mixtures including variouscombinations of GDNF (50 ng/mL), media containing soluble GDNFR (e.g.,PI-PLC/CM derived from NGR-38 cells), and different concentrations ofRet-Fc fusion protein either alone or in various combinations asindicated in FIG. 11. Neuro-2a cells were treated with GDNF, mediacontaining soluble GDNFR, Ret-Fc, or the pre-incubated mixtures. Thecells were then lysed, and the lysates were analyzed for c-Retautophosphorylation by immunoprecipitation using anti-Ret antibody asdescribed in the Experimental Procedures. The immunoprecipitates wereanalyzed by Western blot using an anti-phosphotyrosine antibody.

[0291] The pre-incubated mixture of GDNF and media containing solubleGDNFR induced tyrosine phosphorylation of Ret receptors expressed inNeuro-2a at a level comparable to GDNF-treated NGR-38 control cells(FIG. 11, lanes 7 and 2). The position of the autophosphorylated 170 kDRet bands are marked by a solid arrow. When Ret-Fc fusion protein wasincluded in the pre-incubated GDNF/GDNFR mixture, Ret phosphorylationwas inhibited in a dose dependent manner (FIG. 11, lanes 8-10). Thisindicated that Ret phosphorylation is a result of a GDNF/Ret interactionmediated by GDNFR. In untreated Neuro-2a cells or in cells treated withany combination of GDNF or Ret-Fc fusion protein in the absence ofGDNFR, only background levels of Ret phosphorylation were observed (FIG.11, lanes 3-6).

[0292] GDNF Induces Autophosphorylation of c-RET Expressed in EmbryonicMotor Neurons

[0293] Spinal cord motor neurons are one of the major targets of GDNFaction in vivo (Henderson et al., Science. 266, 1062-1064, 1994; Li etal., Proceedings Of The National Academy Of Sciences, U.S.A. 92,9771-9775, 1995; Oppenheim et al., Nature. 373, 344-346, 1995; Yan etal., Nature. 373, 341-344, 1995; Zurn et al., Neuroreport. 6, 113-118,1995). To test the ability of GDNF to induce Ret autophosphorylation inthese cells, embryonic rat spinal cord motor neurons were treated with(lanes 2 and 4) or without (lanes 1 and 3) 20 ng/mL GDNF followed bylysis of the cells, immunoprecipitation with anti-Ret antibody, andanalysis by Western blotting with anti-phosphotyrosine antibody asdescribed in the Experimental Procedures. In lysates of cells treatedwith GDNF, a band of tyrosine phosphorylated protein with a molecularmass of ˜170 kD was observed (FIG. 12, lane 2). No such signal wasobserved with cells treated with binding buffer alone (FIG. 12, lane 1).When the same Western blot filter was stripped and re-probed withanti-Ret antibody (i.e., the amount of c-Ret protein loaded in each lanewas determined by reprobing the immunoblot with the anti-Ret antibody),bands with the same molecular mass and similar intensities appeared inboth samples (FIG. 12, lanes 3 and 4). The phosphotyrosine band inGDNF-treated cells co-migrates with the Ret protein band, indicatingGDNF stimulated autophosphorylation of Ret. The autophosphorylated Retbands (lanes 1 and 2) and the corresponding protein bands (lanes 3 and4) were marked by a solid arrow.

Discussion

[0294] Polypeptide growth factors elicit biological effects throughbinding to their cognate cell surface receptors. Receptors can begrouped into several classes based on their structure and mechanism ofaction. These classifications include the protein tyrosine kinases(PTKs), the serine/threonine kinases, and the cytokine receptors.Receptor PTK signaling is initiated by a direct interaction with ligand,which induces receptor dimerization or oligomerization that in turnleads to receptor autophosphorylation. The activated receptor thenrecruits and phosphorylates intracellular substrates, initiating acascade of events which culminates in a biological response(Schlessinger and Ullrich, Neuron 9, 383-391, 1992). In contrast, signaltransduction by serine/threonine kinase or cytokine receptors ofteninvolves formation of multi-component receptor complexes in which theligand binding and signal transducing components are distinct. Examplesare the TGF-receptor complex, a serine/threonine kinase receptorconsisting of separate binding (Type II) and signaling (Type I)components and the CNTF family. CNTF, interleukin-6 (IL-6) and leukocyteinhibitory factor (LIF) share the common signaling components, gp130and/or LIFR, in their respective receptor complexes. While the ligandspecificity of these complexes is determined by a specific bindingsubunit to each individual ligand, signal transduction requiresassociation of the initial complex of ligand and ligand binding subunitwith other receptor subunits which cannot bind ligand directly (Ip etal., Cell. 69, 1121-1132, 1992). In the CNTF receptor complex, theligand binding component is CNTF receptor (CNTFR), which like GDNFR, isa GPI-anchored membrane protein. The present invention involves thedescription of the first example of a receptor PTK whoseautophosphorylation is dependent upon association with a separateligand-specific binding component.

[0295] The present study confirms that GDNFR, a GPI-linked membraneprotein that binds to GDNF with high affinity, is required for theefficient association of GDNF with the Ret receptor PTK. In the absenceof GDNFR, GDNF is unable to bind to Ret or stimulate Ret receptorautophosphorylation. In the presence of GDNFR, GDNF associates with Retand rapidly induces Ret autophosphorylation in a dose-dependent manner.GDNFR is able to function in either membrane bound or soluble forms(FIG. 11), as discussed above. GDNF concentrations of 50 pg/mL (1.7 pM)are able activate the Ret tyrosine kinase in cells expressing GDNFR.This is consistent with the dissociation constant (1.5 pM) found for thehigh affinity GDNF binding sites on NGR-38 cells. The rapid induction ofRet phosphorylation by GDNF (detectable one minute after treatment) andthe ability of Ret-Fc to block autophosphorylation suggest that Ret isbeing activated directly rather than as a downstream consequence of thephosphorylation of some other receptor.

[0296] Cross-linking studies support the hypothesis that efficientassociation of Ret with GDNF depends on GDNFR. Cross-linking of GDNF toRet in NGR-38 cells which express high levels of GDNFR is robust, but inparental Neuro-2a cells cross-linked products are barely detectable.Although conclusive identification of all the cross-linked complexes isdifficult, the data clearly demonstrates an association of Ret with GDNFthat is dependent on the presence of GDNFR, and demonstrates that GDNFRis included in some of the cross-linked products. The reason for thepresence of minor cross-linked species in Neuro-2a cells is not clear.While the expression of GDNFR mRNA in Neuro-2a cells could not bedetected by Northern blot, it is possible that GDNFR is expressed atvery low levels in these cells.

[0297] The fact that Ret can be activated by GDNF in cultured ratembryonic spinal cord motor neurons further demonstrates the biologicalrelevance of the Ret/GDNF interaction. These cells are a primary targetof GDNF in vivo, and have been shown to respond to low doses of GDNF invitro (Henderson et al., 1994). Stimulation of Ret phosphorylation wasabolished when the motor neuron cells were pre-treated with PI-PLC (datanot shown), suggesting that the activation of Ret by GDNF requiresGDNFR.

[0298] Although binding of ligand to the receptor extracellular domainis the first step in the activation of other known receptor PTKs, thepresent data has shown that this is not the case for GDNF and Ret. FIG.13 depicts a model for the binding of GDNF to GDNFR and Ret, and theconsequent activation of the Ret PTK in response to GDNF. The initialevent in this process is the binding of disulfide-linked dimeric GDNF toGDNFR in either monomeric or dimeric form. Although there is currentlyno direct evidence for the existence of dimeric GDNFR, when 293T cellswere transfected with GDNFR cDNA, two classes of binding sites appeared.The simplest explanation for this observation is the existence ofmonomeric and dimeric GDNFR, each with its own ligand binding affinity.This is consistent with the finding that GDNF binding affinities areapparently unaffected by the presence of Ret. Since the presentexperiments do not address the question of whether dimeric GDNFR is inequilibrium with its monomer in the absence of GDNF or if dimerizationis induced by GDNF binding, these possibilities are presented asalternate pathways. The complex consisting of dimeric GDNFR and dimericGDNF can bind two molecules of Ret, forming the active signalingcomplex. As for other PTKs, close contact between the intracellularcatalytic domains of two Ret molecules is likely to result in receptorautophosphorylation. This notion that Ret functions by this mechanism issupported by the fact that the MEN2A mutation which causes steady statedimerization of Ret results in constituitive activation of the Retkinase (Santoro et al., 1995).

[0299] Motor neurons have been reported to respond to GDNF with an ED₅₀of as low as 5 fM (Henderson et al., 1994). Although it is difficult tocompare binding affinity with the ED₅₀ for a biological response, it ispossible that very high affinity GDNF binding sites exist on thesecells. Other cells, such as embryonic chick sympathetic neurons, havebeen reported to bind GDNF with a K_(d) of 1-5 nM (Trupp et al., JournalOf Cell Biology. 130, 137-148, 1995). It is unlikely that GDNFR isinvolved in a receptor complex for such low affinity sites, but a weakdirect interaction between GDNF and Ret may be present.

[0300] Expression of c-ret has been observed during embryogenesis inmany cell lineages of the developing central and peripheral nervoussystems, including cells of the enteric nervous system (Pachnis, et al.,Development, 119, 1005-1017, 1993; Tsuzuki et al., 1995). Outside thenervous system, c-ret expression has been detected in the Wolffian duct,ureteric bud epithelium and collecting ducts of the kidney (Pachnis, etal., supra; Tsuzuki et al., 1995). Ret expression has also been detectedin all neuroblastoma cell lines derived from the neural crest (Ikeda etal., 1990) and from surgically resected neuroblastomas (Nagao et al.,1990; Takahashi & Cooper, 1987). GDNF expression has been observed inboth CNS and PNS, as well as in non-neuronal tissues during embryonicdevelopment. The levels of GDNF expression found in many non-neuronaltissues were higher than in the nervous system (Choi-Lundberg and Bohn,Brain Res. Dev. Brain Res. 85, 80-88, 1995). Although expression ofGDNFR has not been extensively studied, primary Northern blot analysisdetected the presence of high levels of the GDNFR mRNA in the liver,brain, and kidney of adult rat and mouse. The similarity of theexpression patterns of ret, GDNF, and GDNFR in developing nervous systemand kidney is consistent with their combined action during development.

[0301] Mammalian kidney development has been postulated to result fromreciprocal interactions between the metanephron and the developingureter, a branch developed from the caudal part of the Wolffian duct(Saxen, Organogenesis of the kidney. Development and Cell Biologyseries, Cambridge University Press, Cambridge, England, 1987). While theexpression of Ret has been found at the ureteric bud but not in thesurrounding mesenchyme in developing embryos, the expression of GDNF wasdetected in the undifferentiated but not adult metanephric cap of thekidney. These observations suggest that an interaction between GDNF andRet is responsible for initiating the development of the uretericstructure. Further support for this hypothesis is provided by targeteddisruptions of the GDNF and ret genes, which result in very similarphenotypic defects in kidney (Schuchardt et al., Nature. 367, 380-383,1994; Sanchez, in press). Another major phenotypic defect observed inboth GDNF (−/−) and ret (−/−) knockout animals is a complete loss of theenteric neurons throughout the digestive tract. Hirschsprung's disease,a genetic disorder characterized by the congenital absence ofparasympathetic innervation in the lower intestinal tract, has also beenlinked to “loss-of-function” mutations in ret (Romeo et al., Nature.367, 377-378, 1994. Edery et al., 1994). A later report (Angrist et al.,Hum. Mol.Genet. 4, 821-830, 1995) indicated that, contrary to earlierobservations, some Hirschsprung's patients do not carry mutations inret. It is now envisioned that such patients may carry mutations inGDNF, GDNFR or some other critical component of this signaling pathway.

Experimental Procedures

[0302] [¹²⁵I]GDNF Binding to Neuro-2a Cells Expressing GDNFR

[0303] Neuro-2a cells (ATCC #CCL 131) were transfected with anexpression plasmid, as described above, using the Calcium PhosphateTransfection System (GIBCO/BRL) according to the manufacturer'sdirections. Transfected cells were selected for expression of theplasmid by growth in 400 μg/mL G418 antibiotic (Sigma). G418 resistantclones were expanded and assayed for GDNFR expression by binding to[¹²⁵I]GDNF (Amersham, Inc., custom iodination, catalog #IMQ1057). Cellsfrom each clone were seeded at a density of 3×10⁴ cells/cm² in duplicatewells of 24-well tissue culture plates (Becton Dickinson) pre-coatedwith polyomithine. Cells were washed once with ice-cold washing buffer(DMEM containing 25 mM HEPES, pH 7.5) and were then incubated with 50 pM[¹²⁵I]GDNF in binding buffer (washing buffer plus 0.2% BSA) at 4° C. forfour hours either in the presence or absence of 500 mM unlabeled GDNF.Cells were then washed four times with ice-cold washing buffer, lysed in1 M NaOH, and the cell-associated radiolabel quantitated in a 1470Wizard Automated Gamma Counter (Wallac Inc.). The amount of GDNFRexpressed by individual clones was estimated by the ratio of [¹²⁵I]GDNFbound to cells in the absence and presence of unlabeled GDNF. Threeclones were chosen as representatives of high, moderate, and low levelexpressors of GDNFR for use in binding experiments. The ratios[¹²⁵I]GDNF bound in the absence and presence of unlabeled GDNF for theseclones were: NGR-38) 16:1, NGR-16) 12.8:1, and NGR-33) 8:1. Equilibriumbinding of [¹²⁵I]GDNF to NGR-38 cells was carried out as described aboveexcept that concentrations of labeled GDNF ranged from 0.5 pM to 1 nM.In all assays, nonspecific binding as estimated by the amount ofradiolabel binding to cells in the presence of 500 nM unlabeled GDNF wassubtracted from binding in the absence of unlabeled GDNF. Binding datawas analyzed by Scatchard plot.

[0304] Chemical Cross-Linking

[0305] Neuro-2a or NGR-38 cells were washed once with phosphate-bufferedsaline (PBS, pH 7.1), then treated for four hours at 4° C. with 1 or 3nM [¹²⁵I]GDNF in binding buffer in the presence or absence of 500 nMunlabeled GDNF. Following binding, cells were washed four times withice-cold washing buffer and incubated at room temperature for 45 minuteswith 1 mM bis suberate (BS³, Pierce) in washing buffer. Thecross-linking reaction was quenched by washing the cells three timeswith Tris-buffered saline (TBS, pH 7.5). The cells were then eitherlysed directly in SDS-PAGE sample buffer (80 mM Tris HCl [pH 6.8], 10%glycerol, 1% SDS, 0.025% bromophenol blue) or in Triton X-100 lysisbuffer (50 mM Hepes, pH 7.5, 1% Triton X-100, 50 mM NaCl, 50 mM NaF, 10mM sodium pyrophosphate, 1% aprotinin (Sigma, Cat.# A-6279), 1 mM PMSF(Sigma, Cat.# P-7626), 0.5 mM Na₃VO₄ (Fisher Cat.# S454-50). The lysateswere clarified by centrifugation, incubated with 5 μg/mL of anti-Retantibody (Santa Cruz Antibody, C-19, Cat. #SC-167), and the resultingimmunocomplexes were collected by precipitation with protein A-SepharoseCL-4B (Pharmacia). The immunoprecipitates were washed three times withthe lysis buffer, once with 0.5% NP-40 containing 50 mM NaCl and 20 mMTris-Cl, pH 7.5, and were then resuspended in SDS-PAGE sample buffer.Both the whole cell lysates and the immunoprecipitates were fractionatedby 7.5% SDS-PAGE with a ratio of Bis:Acrylamide at 1:200.

[0306] Western Blot Analysis

[0307] The autophosphorylation of Ret receptor was examined by Westernblot analysis. Briefly, cells were seeded 24 hours prior to the assay in6-well tissue culture dishes at a density of 1.5×10⁶ cells/well. Cellswere washed once with binding buffer and treated with variousconcentrations of different reagents (including GDNF, PI-PLC, PI-PLC/CM,and Ret-Fc fusion protein), either alone or in combination, in bindingbuffer for various periods of times. Treated cells and untreatedcontrols were lysed in Triton X-100 lysis buffer and immunoprecipitatedwith the anti-Ret antibody (Santa Cruz, C-19, Cat. #SC-167) andprotein-A Sepharose as described above. Immunoprecipitates werefractionated by SDS-PAGE and transferred to nitrocellulose membranes asdescribed by Harlow and Lane (Antibodies: A Laboratory Manual. ColdSpring Harbor Laboratory: Cold Spring Harbor, N.Y., 1988). The membraneswere pre-blocked with 5% BSA (Sigma) and the level of tyrosinephosphorylation of the receptor was determined by blotting the membranewith an anti-phosphotyrosine monoclonal antibody 4G10 (UBI, Cat.#05-321) at room temperature for two hours. The amount of proteinincluded in each lane was determined by stripping and re-probing thesame membrane with the anti-Ret antibody. Finally, the membrane wastreated with chemiluminescence reagents (ECL, Amersham) following themanufacturer's instructions and exposed to X-ray films (Hyperfilm-ELC,Amersham).

[0308] Treatment of Cells with PI-PLC and Generation of PI-PLC TreatedConditioned Media

[0309] In order to release GPI-linked GDNFR from the cell surface, cellswere washed once with washing buffer, then incubated with 1 U/mLphosphatidylinositol specific phospholipase C (PI-PLC, BoehringerMannheim, Cat. #1143069) in binding buffer at 37° C. for 45 minutes. Thecells were then washed three times with washing buffer and furtherprocessed for Ret autophosphorylation assay or cross-linking. Forgeneration of PI-PLC treated conditioned media (PI-PLC/CM), 8×10⁶ cellswere removed from tissue culture dishes by treating the cells with PBScontaining 2 mM of EDTA at 37° C. for 5 to 10 minutes. Cells were washedonce with washing buffer, resuspended in 1 mL of binding buffercontaining 1 U/mL of PI-PLC, and incubated at 37° C. for 45 minutes. Thecells were pelleted, and the PI-PLC/CM was collected.

[0310] Preparation of the Ret-Fc Fusion Protein

[0311] A cDNA encompassing the entire coding region of c-Ret wasisolated from a day 17 rat placenta cDNA library using anoligonucleotide probe corresponding to the first 20 amino acids of themouse c-Ret (Iwamoto et al., 1993; van Heyningen, 1994). The regioncoding for the extracellular domain of the Ret receptor (ending with thelast amino acid, R636) was fused in-frame with the DNA coding for the Fcregion of human IgG (IgG1) and subcloned into the expression vectorpDSR2 as previously described (Bartley et al., Nature. 368, 558-560,1994). The ret-Fc/pDSRa2 plasmid was transfected into Chinese hamsterovary (CHO) cells and the recombinant Ret-Fc fusion protein was purifiedby affinity chromatography using a Ni⁺⁺ column (Qiagen).

[0312] Preparation of Embryonic Rat Spinal Cord Motor Neuron Cultures

[0313] Enriched embryonic rat spinal cord motor neuron cultures wereprepared from entire spinal cords of E15 Sprague-Dawley rat fetuses 24hours before the experiments. The spinal cords were dissected, and themeninges and dorsal root ganglia (DRGs) were removed. The spinal cordswere cut into smaller fragments and digested with papain in L15 medium(Papain Kit, Worthington). The motor neurons, which are larger thanother types of cells included in the dissociated cell suspension, wereenriched using a 6.8% Metrizamide gradient (Camu and Henderson, JNeuroscience. 44, 59-70, 1992). Enriched motor neurons residing at theinterface between the metrizamide cushion and the cell suspension werecollected, washed, and seeded in tissue culture dishes pre-coated withpoly-L-ornithine and laminin at a density of ˜9×10⁴ cells/cm² and werecultured at 37° C.

[0314] Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

[0315] While the present invention has been described in terms ofpreferred embodiments and exemplary nucleic acid and amino acidsequences, it is understood that variations and modifications will occurto those skilled in the art. Therefore, it is intended that the appendedclaims cover all such equivalent variations which come within the scopeof the invention as claimed.

REFERENCES

[0316] Angrist, M., Bolk, S., Thiel, B., Puffenberger, E. G., Hofstra,R. M., Buys, C. H., Cass, D. T., and Chakravarti, A. (1995). Mutationanalysis of the RET receptor tyrosine kinase in Hirschsprung disease.Hum. Mol.Genet.4, 821-830.

[0317] Arenas, E., Trupp, M., Akerud, P., and Ibanez, C. F. (1995). GDNFPrevents degeneration and promotes the phenotype of brain noradrenergicneurons in vivo. Neuron 15, 1465-1473.

[0318] Aruffo, A. and Seed, B. (1987). Molecular cloning of a CD28 cDNAby a high-efficiency COS cell expression system. Proceedings Of TheNational Academy Of Sciences Of The United States Of America. 84,8573-8577.

[0319] Bartley, T. D., Hunt, R. W., Welcher, A. A., Boyle, W. J.,Parker, V. P., Lindberg, R. A., Lu, H. S., Colombero, A. M., Elliott, R.L., Guthrie, B. A., Holst, P. L., Skrine, J. D., Toso, R. J., Zhang, M.,Fernandez, E., Trail, G., Varnum, B., Yarden, Y., Hunter, T., and Fox,G. M. (1994). B61 is a Ligand for the ECK Receptor protein-tyrosinekinase. Nature. 368, 558-560.

[0320] Beck, K. D., Valverde, J., Alexi, T., Poulsen, K., Moffat, B.,Vandlen, R. A., Rosenthal, A., and Hefti, F. (1995). Mesencephalicdopaminergic neurons protected by GDNF from axotomy-induced degenerationin the adult brain. Nature. 373, 339-341.

[0321] Camu, W. and Henderson, C. (1992). Purification of embryonic ratmotoneurons by panning on a monoclonal antibody to the low-affinity NGFreceptor. J Neuroscience. 44, 59-70.

[0322] Choi-Lundberg, D. L. and Bohn, M. C. (1995). Ontogeny anddistribution of glial cell line-derived neurotrophic factor (GDNF) mRNAin rat. Brain Res. Dev. Brain Res. 85, 80-88.

[0323] Davis, S., Aldrich, T. H., Valenzuela, D. M., Wong, V. V., Furth,M. E., Squinto, S. P., and Yancopoulos, G. D. (1991). The receptor forciliary neurotrophic factor. Science. 253, 59-63.

[0324] Donis-Keller, H., Dou, S., Chi, D., Carlson, K., Toshima, K.,Lairmore, T., Howe, J., Moley, J., Goodfellow, P. and Wells, S. (1993).Mutations in the ret proto-oncogene are associated with MEN 2A and FMTC.Hum. Molec. Genet. 2, 851-856.

[0325] Ebendal, T., Tomac, A., Hoffer, B. J., and Olson, L. (1995).Glial cell line-derived neurotrophic factor stimulates fiber formationand survival in cultured neurons from peripheral autonomic ganglia.Journal Of Neuroscience Research. 40, 276-284.

[0326] Economides, A. N., Ravetch, J. V., Yancopoulos, G. D., and Stahl,N. (1995). Designer cytokines: targeting actions to cells of choice.Science 270, 1351-1353.

[0327] Edery, P., Lyonnet, S., Mulligan, L., Pelet, A., Dow, E., Abel,L., Holder, S., Nihoul-Fekete, C., Ponder, B. and Munnich, A. (1994).Mutations of the ret proto-oncogene in Hirschsprug's disease. Nature.367, 378-380.

[0328] Gearing, D. P., King, J. A., Gough, N. M., and Nicola, N. A.(1989). Expression cloning of a receptor for humangranulocyte-macrophage colony-stimulating factor. EMBO Journal 8,3667-3676.

[0329] Henderson, C. E., Phillips, H. S., Pollock, R. A., Davies, A. M.,Lemeulle, C., Armanini, M., Simpson, L. C., Moffet, B., Vandlen, R. A.,Koliatsos, V. E., and et al (1994). GDNF: a potent survival factor formotoneurons present in peripheral nerve and muscle. Science. 266,1062-1064.

[0330] Hoffer, B. J., Hoffman, A., Bowenkamp, K., Huettl, P., Hudson,J., Martin, D., Lin, L. F., and Gerhardt, G. A. (1994). Glial cellline-derived neurotrophic factor reverses toxin-induced injury tomidbrain dopaminergic neurons in vivo. Neuroscience Letters. 182,107-111.

[0331] Hofstra, R., Landsvater, R., Ceccherini, I., Stulp, R.,Stelwagen, T., Luo, Y., Pasini, B., Hoppener, J., van Amstel, H., Romeo,G., Lips, C. and Buys, C. (1994). A mutation in the ret proto-oncogeneassociated with multipleendocrine neoplasia type 2B and sporadicmedullary thyroid carcinoma. Nature. 367, 375-376.

[0332] Ikeda, I., Ishizaka, Y., Tahira, T., Suzuki, T., Onda, M.,Sugimura, T., and Nagao, M. (1990). Specific expression of the retproto-oncogene in human neuroblastoma cell lines. Oncogene. 5,1291-1296.

[0333] Ip, N. Y., Nye, S. H., Boulton, T. G., Davis, S., Yasukawa, K.,Kishimoto, T., Anderson, D. J., and et al (1992). CNTF and LIF act onneuronal cells via shared signaling pathways that involve the IL-6signal transducing receptor component gp130. Cell. 69, 1121-1132.

[0334] Iwamoto, T., Taniguchi, M., Asia, N., Ohkusu, K., Nakashima, I.and Takahashi, M. (1993). cDNA cloning of mouse ret proto-oncongene andits sequence similarity to the cadherin superfamily. Oncogene. 8,1087-1091.

[0335] Jing, S. Q., Spencer, T., Miller, K., Hopkins, C., andTrowbridge, I. S. (1990). Role of the human transferrin receptorcytoplasmic domain in endocytosis: localization of a specific signalsequence for internalization. Journal Of Cell Biology. 110, 283-294.

[0336] Kearns, C. M. and Gash, D. M. (1995). GDNF protects nigraldopamine neurons against 6-hydroxydopamine in vivo. Brain Research. 672,104-111.

[0337] Kozak, M. (1987). An analysis of 5′-noncoding sequences from 699vertebrate messenger RNAs. Nucleic Acids Research. 15, 8125-8148.

[0338] Li, L., Wu, W., Lin, L. F., Lei, M., Oppenheim, R. W., andHouenou, L. J. (1995). Rescue of adult mouse motoneurons frominjury-induced cell death by glial cell line-derived neurotrophicfactor. Proceedings Of The National Academy Of Sciences Of The UnitedStates Of America. 92, 9771-9775.

[0339] Lin, L-F. H., Doherty, D. H., Lile, J. D., Bektesh, S., andCollins, F. (1993). GDNF: a glial cell line-derived neurotrophic factorfor midbrain dopaminergic neurons. Science. 260, 1130-1132.

[0340] Louis, J. C., Magal, E., and Varon, S. (1992). Receptor-mediatedtoxicity of norepinephrine on cultured catecholaminergic neurons of therat brain stem. Journal Of Pharmacology And Experimental Therapeutics.262, 1274-1283.

[0341] Mount, H. T., Dean, D. O., Alberch, J., Dreyfus, C. F., andBlack, I. B. (1995). Glial cell line-derived neurotrophic factorpromotes the survival and morphologic differentiation of Purkinje cells.Proceedings Of The National Academy Of Sciences Of The United States OfAmerica. 92, 9092-9096.

[0342] Mulligan, L., Kwok, J., Healey, C., Elsdon, M., Eng, C., Gardner,E., Love, D., Mole, S., Moore, J., Papi, L., Ponder, M., Telenius, H.,Tunnacliffe, A. and Ponder, A. (1993). Germ-line mutations of the retproto-oncongene in mutiple endocrine neoplasia type 2A. Nature. 363,458-460.

[0343] Oppenheim, R. W., Houenou, L. J., Johnson, J. E., Lin, L. F., Li,L., Lo, A. C., Newsome, A. L., Prevette, D. M., and Wang, S. (1995).Developing motor neurons rescued from programmed and axotomy-inducedcell death by GDNF. Nature. 373, 344-346.

[0344] Pachnis, V., Mankoo, B., and Costantini, F. (1993). Expression ofthe c-ret proto-oncogene during mouse embryogenesis. Development, 119,1005-1017.

[0345] Pearson, W. R. and Lipman, D. J. (1988). Improved tools forbiological sequence comparison. Proceedings Of The National Academy OfSciences Of The United States Of America. 85, 2444-2448.

[0346] Poulsen, K. T., Armanini, M. P., Klein, R. D., Hynes, M. A.,Phillips, H. S., and Rosenthal, A. (1994). TGF beta 2 and TGF beta 3 arepotent survival factors for midbrain dopaminergic neurons. Neuron. 13,1245-1252.

[0347] Romeo, G., Patrizia, R, Luo, Y., Barone, V., Seri, M.,Ceccherini, I., Pasini, B., Bocciardi, R., Lerone, M., Kaariainen, H.and Maartucciello, G. (1994). Point mutations affecting the tyrosinekinase domain of the ret proto-oncogene in Hirschsprung's disease.Nature. 367, 377-378.

[0348] Santoro, M., Carlomagno, F., Romeo, A., Bottaro, D., Dathan, N.,Grieco, M., Fusco, A., Vecchio, G., Matoskova, B., Kraus, M. and DiFiore, P. (1995). Activation of ret as a dominant transforming gene bygermline mutations of MEN2A and MEN2B. Science. 267, 381-383.

[0349] Sauer, H., Rosenblad, C., and Bjoerklund, A. (1995). Glial cellline-derived neurotrophic factor but not transforming growth factor beta3 prevents delayed degeneration of nigral dopaminergic neurons followingstriatal 6-hydroxydopamine lesion. Proceedings Of The National AcademyOf Sciences Of The United States Of America. 92, 8935-8939.

[0350] Saxen, L. (1987). Organogenesis of the kidney. Development andCell Biology series, Cambridge University Press, Cambridge, England.

[0351] Schaar, D. G., Sieber, B. A., Dreyfus, C. F., and Black, I. B.(1993). Regional and cell-specific expression of GDNF in rat brain.Experimental Neurology. 124, 368-371.

[0352] Schaar, D. G., Sieber, B. A., Sherwood, A. C., Dean, D., Mendoza,G., Ramakrishnan, L., Dreyfus, C. F., and Black, I. B. (1994). Multipleastrocyte transcripts encode nigral trophic factors in rat and human.Experimental Neurology. 130, 387-393.

[0353] Schlessinger, J. and Ullrich, A. (1992). Growth factor signalingby receptor tyrosine kinases. Neuron 9, 383-391.

[0354] Schuchardt, A., D'Agati, V., Larsson-Blomberg, L., Costantini, F.and Pachnis, V. (1994). Defects in the kidney and enteric nervous systemof mice lacking the tyrosine kinase receptor ret. Nature. 367, 380-383.

[0355] Segarini, P. R., Ziman, J. M., Kane, C. J., and Dasch, J. R.(1992). Two novel patterns of transforming growth factor beta (TGF-beta)binding to cell surface proteins are dependent upon the binding ofTGF-beta 1 and indicate a mechanism of positive cooperativity. JournalOf Biological Chemistry. 267, 1048-1053.

[0356] Springer, J. E., Mu, X., Bergmann, L. W., and Trojanowski, J. Q.(1994). Expression of GDNF mRNA in rat and human nervous tissue.Experimental Neurology. 127, 167-170.

[0357] Stroemberg, I., Bjoerklund, L., Johansson, M., Tomac, A.,Collins, F., Olson, L., Hoffer, B., and Humpel, C. (1993). Glial cellline-derived neurotrophic factor is expressed in the developing but notadult striatum and stimulates developing dopamine neurons in vivo.Experimental Neurology. 124, 401-412.

[0358] Takahashi, M., Ritz, J. and Cooper, G. (1985). Activation of anovel human tranforming gene, ret, by DNA rearrangement. Cell. 42,581-588.

[0359] Takahashi, M. and Cooper, G. (1987). Ret trasnforming geneencodes a fusion protein homologous to tyrosine kinases. Mol. Cell.Biol., 7, 1378-1385.

[0360] Takebe, Y., Seiki, M., Fujisawa, J., Hoy, P., Yokota, K., Arai,K., Yoshida, M., and Arai, N. (1988). SRa promoter: an efficient andversatile mammalian cDNA expression system composed of the simian virus40 early promoter and the R-U5 segment of human T-cell leukemia virustype 1 long terminal repeat. Mol. Cell. Biol. 8, 466-472.

[0361] Tomac, A., Lindqvist, E., Lin, L. F., Ogren, S. O., Young, D.,Hoffer, B. J., and Olson, L. (1995a). Protection and repair of thenigrostriatal dopaminergic system by GDNF in vivo. Nature. 373, 335-339.

[0362] Tomac, A., Widenfalk, J., Lin, L. F., Kohno, T., Ebendal, T.,Hoffer, B. J., and Olson, L. (1995b). Retrograde axonal transport ofglial cell line-derived neurotrophic factor in the adult nigrostriatalsystem suggests a trophic role in the adult. Proceedings Of The NationalAcademy Of Sciences Of The United States Of America. 92, 8274-8278.

[0363] Trupp, M., Ryden, M., Joervall, H., Funakoshi, H., Timmusk, T.,Arenas, E., and Ibanez, C. F. (1995). Peripheral expression andbiological activities of GDNF, a new neurotrophic factor for avian andmammalian peripheral neurons. Journal Of Cell Biology. 130, 137-148.

[0364] Tsuzuki, T., Takahashi, M., Asai, N., Iwashita, T., Matsuyama, M.and Asai, J. (1995). Spatial and temporal expression of the retproto-oncongene product in embryonic, infant and adult rat tissues.Oncogene, 10, 191-198.

[0365] Ullrich, A and Schlessinger, J. (1990). Signal transduction byreceptors with tyrosine kinase activity. Cell, 61, 203-211.

[0366] van der Geer, P., Hunter, T., and Lindberg, R. A. (1994).Receptor protein-tyrosine kinases and their signal transductionpathways. 10, 251-337.

[0367] van Heyningen, V. (1994). One gene-four syndromes. Nature, 367,319-320.

[0368] von Heijne, G. (1986). A new method for predicting signalsequence cleavage sites. Nucleic Acids Research. 14, 4683-4690.

[0369] Yan, Q., Matheson, C., and Lopez, O. T. (1995). In vivoneurotrophic effects of GDNF on neonatal and adult facial motor neurons.Nature. 373, 341-344.

[0370] Zurn, A. D., Baetge, E. E., Hammang, J. P., Tan, S. A., andAebischer, P. (1994). Glial cell line-derived neurotrophic factor(GDNF), a new neurotrophic factor for motoneurones. Neuroreport. 6,113-118.

1 47 1 2568 DNA HUMAN CDS (540)..(1934) 1 aatctggcct cggaacacgccattctccgc gccgcttcca ataaccacta acatccctaa 60 cgagcatccg agccgagggctctgctcgga aatcgtcctg gcccaactcg gcccttcgag 120 ctctcgaaga ttaccgcatctatttttttt ttcttttttt tcttttccta gcgcagataa 180 agtgagcccg gaaagggaaggagggggcgg ggacaccatt gccctgaaag aataaataag 240 taaataaaca aactggctcctcgccgcagc tggacgcggt cggttgagtc caggttgggt 300 cggacctgaa cccctaaaagcggaaccgcc tcccgccctc gccatcccgg agctgagtcg 360 ccggcggcgg tggctgctgccagacccgga gtttcctctt tcactggatg gagctgaact 420 ttgggcggcc agagcagcacagctgtccgg ggatcgctgc acgctgagct ccctcggcaa 480 gacccagcgg cggctcgggatttttttggg ggggcgggga ccagccccgc gccggcacc 539 atg ttc ctg gcg acc ctgtac ttc gcg ctg ccg ctc ttg gac ttg ctc 587 Met Phe Leu Ala Thr Leu TyrPhe Ala Leu Pro Leu Leu Asp Leu Leu 1 5 10 15 ctg tcg gcc gaa gtg agcggc gga gac cgc ctg gat tgc gtg aaa gcc 635 Leu Ser Ala Glu Val Ser GlyGly Asp Arg Leu Asp Cys Val Lys Ala 20 25 30 agt gat cag tgc ctg aag gagcag agc tgc agc acc aag tac cgc acg 683 Ser Asp Gln Cys Leu Lys Glu GlnSer Cys Ser Thr Lys Tyr Arg Thr 35 40 45 cta agg cag tgc gtg gcg ggc aaggag acc aac ttc agc ctg gca tcc 731 Leu Arg Gln Cys Val Ala Gly Lys GluThr Asn Phe Ser Leu Ala Ser 50 55 60 ggc ctg gag gcc aag gat gag tgc cgcagc gcc atg gag gcc ctg aag 779 Gly Leu Glu Ala Lys Asp Glu Cys Arg SerAla Met Glu Ala Leu Lys 65 70 75 80 cag aag tcg ctc tac aac tgc cgc tgcaag cgg ggt atg aag aag gag 827 Gln Lys Ser Leu Tyr Asn Cys Arg Cys LysArg Gly Met Lys Lys Glu 85 90 95 aag aac tgc ctg cgc att tac tgg agc atgtac cag agc ctg cag gga 875 Lys Asn Cys Leu Arg Ile Tyr Trp Ser Met TyrGln Ser Leu Gln Gly 100 105 110 aat gat ctg ctg gag gat tcc cca tat gaacca gtt aac agc aga ttg 923 Asn Asp Leu Leu Glu Asp Ser Pro Tyr Glu ProVal Asn Ser Arg Leu 115 120 125 tca gat ata ttc cgg gtg gtc cca ttc atatca gat gtt ttt cag caa 971 Ser Asp Ile Phe Arg Val Val Pro Phe Ile SerAsp Val Phe Gln Gln 130 135 140 gtg gag cac att ccc aaa ggg aac aac tgcctg gat gca gcg aag gcc 1019 Val Glu His Ile Pro Lys Gly Asn Asn Cys LeuAsp Ala Ala Lys Ala 145 150 155 160 tgc aac ctc gac gac att tgc aag aagtac agg tcg gcg tac atc acc 1067 Cys Asn Leu Asp Asp Ile Cys Lys Lys TyrArg Ser Ala Tyr Ile Thr 165 170 175 ccg tgc acc acc agc gtg tcc aac gatgtc tgc aac cgc cgc aag tgc 1115 Pro Cys Thr Thr Ser Val Ser Asn Asp ValCys Asn Arg Arg Lys Cys 180 185 190 cac aag gcc ctc cgg cag ttc ttt gacaag gtc ccg gcc aag cac agc 1163 His Lys Ala Leu Arg Gln Phe Phe Asp LysVal Pro Ala Lys His Ser 195 200 205 tac gga atg ctc ttc tgc tcc tgc cgggac atc gcc tgc aca gag cgg 1211 Tyr Gly Met Leu Phe Cys Ser Cys Arg AspIle Ala Cys Thr Glu Arg 210 215 220 agg cga cag acc atc gtg cct gtg tgctcc tat gaa gag agg gag aag 1259 Arg Arg Gln Thr Ile Val Pro Val Cys SerTyr Glu Glu Arg Glu Lys 225 230 235 240 ccc aac tgt ttg aat ttg cag gactcc tgc aag acg aat tac atc tgc 1307 Pro Asn Cys Leu Asn Leu Gln Asp SerCys Lys Thr Asn Tyr Ile Cys 245 250 255 aga tct cgc ctt gcg gat ttt tttacc aac tgc cag cca gag tca agg 1355 Arg Ser Arg Leu Ala Asp Phe Phe ThrAsn Cys Gln Pro Glu Ser Arg 260 265 270 tct gtc agc agc tgt cta aag gaaaac tac gct gac tgc ctc ctc gcc 1403 Ser Val Ser Ser Cys Leu Lys Glu AsnTyr Ala Asp Cys Leu Leu Ala 275 280 285 tac tcg ggg ctt att ggc aca gtcatg acc ccc aac tac ata gac tcc 1451 Tyr Ser Gly Leu Ile Gly Thr Val MetThr Pro Asn Tyr Ile Asp Ser 290 295 300 agt agc ctc agt gtg gcc cca tggtgt gac tgc agc aac agt ggg aac 1499 Ser Ser Leu Ser Val Ala Pro Trp CysAsp Cys Ser Asn Ser Gly Asn 305 310 315 320 gac cta gaa gag tgc ttg aaattt ttg aat ttc ttc aag gac aat aca 1547 Asp Leu Glu Glu Cys Leu Lys PheLeu Asn Phe Phe Lys Asp Asn Thr 325 330 335 tgt ctt aaa aat gca att caagcc ttt ggc aat ggc tcc gat gtg acc 1595 Cys Leu Lys Asn Ala Ile Gln AlaPhe Gly Asn Gly Ser Asp Val Thr 340 345 350 gtg tgg cag cca gcc ttc ccagta cag acc acc act gcc act acc acc 1643 Val Trp Gln Pro Ala Phe Pro ValGln Thr Thr Thr Ala Thr Thr Thr 355 360 365 act gcc ctc cgg gtt aag aacaag ccc ctg ggg cca gca ggg tct gag 1691 Thr Ala Leu Arg Val Lys Asn LysPro Leu Gly Pro Ala Gly Ser Glu 370 375 380 aat gaa att ccc act cat gttttg cca ccg tgt gca aat tta cag gca 1739 Asn Glu Ile Pro Thr His Val LeuPro Pro Cys Ala Asn Leu Gln Ala 385 390 395 400 cag aag ctg aaa tcc aatgtg tcg ggc aat aca cac ctc tgt att tcc 1787 Gln Lys Leu Lys Ser Asn ValSer Gly Asn Thr His Leu Cys Ile Ser 405 410 415 aat ggt aat tat gaa aaagaa ggt ctc ggt gct tcc agc cac ata acc 1835 Asn Gly Asn Tyr Glu Lys GluGly Leu Gly Ala Ser Ser His Ile Thr 420 425 430 aca aaa tca atg gct gctcct cca agc tgt ggt ctg agc cca ctg ctg 1883 Thr Lys Ser Met Ala Ala ProPro Ser Cys Gly Leu Ser Pro Leu Leu 435 440 445 gtc ctg gtg gta acc gctctg tcc acc cta tta tct tta aca gaa aca 1931 Val Leu Val Val Thr Ala LeuSer Thr Leu Leu Ser Leu Thr Glu Thr 450 455 460 tca tagctgcattaaaaaaatac aatatggaca tgtaaaaaga caaaaaccaa 1984 Ser 465 gttatctgtttcctgttctc ttgtatagct gaaattccag tttaggagct cagttgagaa 2044 acagttccattcaactggaa catttttttt tttncctttt aagaaagctt cttgtgatcc 2104 ttnggggcttctgtgaaaaa cctgatgcag tgctccatcc aaactcagaa ggctttggga 2164 tatgctgtattttaaaggga cagtttgtaa cttgggctgt aaagcaaact ggggctgtgt 2224 tttcgatgatgatgatnatc atgatnatga tnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2284 nnnnnnnnnngattttaaca gttttacttc tggcctttcc tagctagaga aggagttaat 2344 atttctaaggtaactcccat atctccttta atgacattga tttctaatga tataaatttc 2404 agcctacattgatgccaagc ttttttgcca caaagaagat tcttaccaag agtgggcttt 2464 gtggaaacagctggtactga tgttcacctt tatatatgta ctagcatttt ccacgctgat 2524 gtttatgtactgtaaacagt tctgcactct tgtacaaaag aaaa 2568 2 465 PRT HUMAN misc_feature(2078)..(2078) N in position 2078 indicates a position of divergencebetween different receptor clones 2 Met Phe Leu Ala Thr Leu Tyr Phe AlaLeu Pro Leu Leu Asp Leu Leu 1 5 10 15 Leu Ser Ala Glu Val Ser Gly GlyAsp Arg Leu Asp Cys Val Lys Ala 20 25 30 Ser Asp Gln Cys Leu Lys Glu GlnSer Cys Ser Thr Lys Tyr Arg Thr 35 40 45 Leu Arg Gln Cys Val Ala Gly LysGlu Thr Asn Phe Ser Leu Ala Ser 50 55 60 Gly Leu Glu Ala Lys Asp Glu CysArg Ser Ala Met Glu Ala Leu Lys 65 70 75 80 Gln Lys Ser Leu Tyr Asn CysArg Cys Lys Arg Gly Met Lys Lys Glu 85 90 95 Lys Asn Cys Leu Arg Ile TyrTrp Ser Met Tyr Gln Ser Leu Gln Gly 100 105 110 Asn Asp Leu Leu Glu AspSer Pro Tyr Glu Pro Val Asn Ser Arg Leu 115 120 125 Ser Asp Ile Phe ArgVal Val Pro Phe Ile Ser Asp Val Phe Gln Gln 130 135 140 Val Glu His IlePro Lys Gly Asn Asn Cys Leu Asp Ala Ala Lys Ala 145 150 155 160 Cys AsnLeu Asp Asp Ile Cys Lys Lys Tyr Arg Ser Ala Tyr Ile Thr 165 170 175 ProCys Thr Thr Ser Val Ser Asn Asp Val Cys Asn Arg Arg Lys Cys 180 185 190His Lys Ala Leu Arg Gln Phe Phe Asp Lys Val Pro Ala Lys His Ser 195 200205 Tyr Gly Met Leu Phe Cys Ser Cys Arg Asp Ile Ala Cys Thr Glu Arg 210215 220 Arg Arg Gln Thr Ile Val Pro Val Cys Ser Tyr Glu Glu Arg Glu Lys225 230 235 240 Pro Asn Cys Leu Asn Leu Gln Asp Ser Cys Lys Thr Asn TyrIle Cys 245 250 255 Arg Ser Arg Leu Ala Asp Phe Phe Thr Asn Cys Gln ProGlu Ser Arg 260 265 270 Ser Val Ser Ser Cys Leu Lys Glu Asn Tyr Ala AspCys Leu Leu Ala 275 280 285 Tyr Ser Gly Leu Ile Gly Thr Val Met Thr ProAsn Tyr Ile Asp Ser 290 295 300 Ser Ser Leu Ser Val Ala Pro Trp Cys AspCys Ser Asn Ser Gly Asn 305 310 315 320 Asp Leu Glu Glu Cys Leu Lys PheLeu Asn Phe Phe Lys Asp Asn Thr 325 330 335 Cys Leu Lys Asn Ala Ile GlnAla Phe Gly Asn Gly Ser Asp Val Thr 340 345 350 Val Trp Gln Pro Ala PhePro Val Gln Thr Thr Thr Ala Thr Thr Thr 355 360 365 Thr Ala Leu Arg ValLys Asn Lys Pro Leu Gly Pro Ala Gly Ser Glu 370 375 380 Asn Glu Ile ProThr His Val Leu Pro Pro Cys Ala Asn Leu Gln Ala 385 390 395 400 Gln LysLeu Lys Ser Asn Val Ser Gly Asn Thr His Leu Cys Ile Ser 405 410 415 AsnGly Asn Tyr Glu Lys Glu Gly Leu Gly Ala Ser Ser His Ile Thr 420 425 430Thr Lys Ser Met Ala Ala Pro Pro Ser Cys Gly Leu Ser Pro Leu Leu 435 440445 Val Leu Val Val Thr Ala Leu Ser Thr Leu Leu Ser Leu Thr Glu Thr 450455 460 Ser 465 3 2138 DNA RAT CDS (302)..(1705) 3 agctcgctct cccggggcagtggtgtggat gcaccggagt tcgggcgctg ggcaagttgg 60 gtcggaactg aacccctgaaagcgggtccg cctcccgccc tcgcgcccgc ccggatctga 120 gtcgctggcg gcggtgggcggcagagcgac ggggagtctg ctctcaccct ggatggagct 180 gaactttgag tggccagaggagcgcagtcg cccggggatc gctgcacgct gagctctctc 240 cccgagaccg ggcggcggctttggattttg ggggggcggg gaccagctgc gcggcggcac 300 c atg ttc cta gcc actctg tac ttc gcg ctg cca ctc ctg gat ttg ctg 349 Met Phe Leu Ala Thr LeuTyr Phe Ala Leu Pro Leu Leu Asp Leu Leu 1 5 10 15 atg tcc gcc gag gtgagt ggt gga gac cgt ctg gac tgt gtg aaa gcc 397 Met Ser Ala Glu Val SerGly Gly Asp Arg Leu Asp Cys Val Lys Ala 20 25 30 agc gat cag tgc ctg aaggaa cag agc tgc agc acc aag tac cgc aca 445 Ser Asp Gln Cys Leu Lys GluGln Ser Cys Ser Thr Lys Tyr Arg Thr 35 40 45 cta agg cag tgc gtg gcg ggcaag gaa acc aac ttc agc ctg aca tcc 493 Leu Arg Gln Cys Val Ala Gly LysGlu Thr Asn Phe Ser Leu Thr Ser 50 55 60 ggc ctt gag gcc aag gat gag tgccgt agc gcc atg gag gcc ttg aag 541 Gly Leu Glu Ala Lys Asp Glu Cys ArgSer Ala Met Glu Ala Leu Lys 65 70 75 80 cag aag tct ctg tac aac tgc cgctgc aag cgg ggc atg aag aaa gag 589 Gln Lys Ser Leu Tyr Asn Cys Arg CysLys Arg Gly Met Lys Lys Glu 85 90 95 aag aat tgt ctg cgt atc tac tgg agcatg tac cag agc ctg cag gga 637 Lys Asn Cys Leu Arg Ile Tyr Trp Ser MetTyr Gln Ser Leu Gln Gly 100 105 110 aat gac ctc ctg gaa gat tcc ccg tatgag ccg gtt aac agc agg ttg 685 Asn Asp Leu Leu Glu Asp Ser Pro Tyr GluPro Val Asn Ser Arg Leu 115 120 125 tca gat ata ttc cgg gca gtc ccg ttcata tca gat gtt ttc cag caa 733 Ser Asp Ile Phe Arg Ala Val Pro Phe IleSer Asp Val Phe Gln Gln 130 135 140 gtg gaa cac att tcc aaa ggg aac aactgc ctg gac gca gcc aag gcc 781 Val Glu His Ile Ser Lys Gly Asn Asn CysLeu Asp Ala Ala Lys Ala 145 150 155 160 tgc aac ctg gac gac acc tgt aagaag tac agg tcg gcc tac atc acc 829 Cys Asn Leu Asp Asp Thr Cys Lys LysTyr Arg Ser Ala Tyr Ile Thr 165 170 175 ccc tgc acc acc agc atg tcc aacgag gtc tgc aac cgc cgt aag tgc 877 Pro Cys Thr Thr Ser Met Ser Asn GluVal Cys Asn Arg Arg Lys Cys 180 185 190 cac aag gcc ctc agg cag ttc ttcgac aag gtt ccg gcc aag cac agc 925 His Lys Ala Leu Arg Gln Phe Phe AspLys Val Pro Ala Lys His Ser 195 200 205 tac ggg atg ctc ttc tgc tcc tgccgg gac atc gcc tgc acc gag cgg 973 Tyr Gly Met Leu Phe Cys Ser Cys ArgAsp Ile Ala Cys Thr Glu Arg 210 215 220 cgg cga cag act atc gtc ccc gtgtgc tcc tat gaa gaa cga gag agg 1021 Arg Arg Gln Thr Ile Val Pro Val CysSer Tyr Glu Glu Arg Glu Arg 225 230 235 240 ccc aac tgc ctg agt ctg caagac tcc tgc aag acc aat tac atc tgc 1069 Pro Asn Cys Leu Ser Leu Gln AspSer Cys Lys Thr Asn Tyr Ile Cys 245 250 255 aga tct cgc ctt gca gat tttttt acc aac tgc cag cca gag tca agg 1117 Arg Ser Arg Leu Ala Asp Phe PheThr Asn Cys Gln Pro Glu Ser Arg 260 265 270 tct gtc agc aac tgt ctt aaggag aac tac gca gac tgc ctc ctg gcc 1165 Ser Val Ser Asn Cys Leu Lys GluAsn Tyr Ala Asp Cys Leu Leu Ala 275 280 285 tac tcg gga ctg att ggc acagtc atg act ccc aac tac gta gac tcc 1213 Tyr Ser Gly Leu Ile Gly Thr ValMet Thr Pro Asn Tyr Val Asp Ser 290 295 300 agc agc ctc agc gtg gca ccatgg tgt gac tgc agc aac agc ggc aat 1261 Ser Ser Leu Ser Val Ala Pro TrpCys Asp Cys Ser Asn Ser Gly Asn 305 310 315 320 gac ctg gaa gac tgc ttgaaa ttt ctg aat ttt ttt aag gac aat act 1309 Asp Leu Glu Asp Cys Leu LysPhe Leu Asn Phe Phe Lys Asp Asn Thr 325 330 335 tgt ctc aaa aat gca attcaa gcc ttt ggc aat ggc tca gat gtg acc 1357 Cys Leu Lys Asn Ala Ile GlnAla Phe Gly Asn Gly Ser Asp Val Thr 340 345 350 atg tgg cag cca gcc cctcca gtc cag acc acc act gcc acc act acc 1405 Met Trp Gln Pro Ala Pro ProVal Gln Thr Thr Thr Ala Thr Thr Thr 355 360 365 act gcc ttc cgg gtc aagaac aag cct ctg ggg cca gca ggg tct gag 1453 Thr Ala Phe Arg Val Lys AsnLys Pro Leu Gly Pro Ala Gly Ser Glu 370 375 380 aat gag atc ccc aca cacgtt tta cca ccc tgt gcg aat ttg cag gct 1501 Asn Glu Ile Pro Thr His ValLeu Pro Pro Cys Ala Asn Leu Gln Ala 385 390 395 400 cag aag ctg aaa tccaat gtg tcg ggt agc aca cac ctc tgt ctt tct 1549 Gln Lys Leu Lys Ser AsnVal Ser Gly Ser Thr His Leu Cys Leu Ser 405 410 415 gat agt gat ttc ggaaag gat ggt ctc gct ggt gcc tcc agc cac ata 1597 Asp Ser Asp Phe Gly LysAsp Gly Leu Ala Gly Ala Ser Ser His Ile 420 425 430 acc aca aaa tca atggct gct cct ccc agc tgc agt ctg agc tca ctg 1645 Thr Thr Lys Ser Met AlaAla Pro Pro Ser Cys Ser Leu Ser Ser Leu 435 440 445 ccg gtg ctg atg ctcacc gcc ctt gct gcc ctg tta tct gta tcg ttg 1693 Pro Val Leu Met Leu ThrAla Leu Ala Ala Leu Leu Ser Val Ser Leu 450 455 460 gca gaa acg tcgtagctgcatc cgggaaaaca gtatgaaaag acaaaagaga 1745 Ala Glu Thr Ser 465accaagtatt ctgtccctgt cctcttgtat atctgaaaat ccagttttaa aagctccgtt 1805gagaagcagt ttcacccaac tggaactctt tccttgtttt taagaaagct tgtggccctc 1865aggggcttct gttgaagaac tgctacaggg ctaattccaa acccataagg ctctggggcg 1925tggtgcggct taaggggacc atttgcacca tgtaaagcaa gctgggctta tcatgtgttt 1985gatggtgagg atggtagtgg tgatgatgat ggtaatttta acagcttgaa ccctgttctc 2045tctactggtt aggaacagga gatactattg ataaagattc ttccatgtct tactcagcag 2105cattgccttc tgaagacagg cccgcagccg tcg 2138 4 468 PRT RAT 4 Met Phe LeuAla Thr Leu Tyr Phe Ala Leu Pro Leu Leu Asp Leu Leu 1 5 10 15 Met SerAla Glu Val Ser Gly Gly Asp Arg Leu Asp Cys Val Lys Ala 20 25 30 Ser AspGln Cys Leu Lys Glu Gln Ser Cys Ser Thr Lys Tyr Arg Thr 35 40 45 Leu ArgGln Cys Val Ala Gly Lys Glu Thr Asn Phe Ser Leu Thr Ser 50 55 60 Gly LeuGlu Ala Lys Asp Glu Cys Arg Ser Ala Met Glu Ala Leu Lys 65 70 75 80 GlnLys Ser Leu Tyr Asn Cys Arg Cys Lys Arg Gly Met Lys Lys Glu 85 90 95 LysAsn Cys Leu Arg Ile Tyr Trp Ser Met Tyr Gln Ser Leu Gln Gly 100 105 110Asn Asp Leu Leu Glu Asp Ser Pro Tyr Glu Pro Val Asn Ser Arg Leu 115 120125 Ser Asp Ile Phe Arg Ala Val Pro Phe Ile Ser Asp Val Phe Gln Gln 130135 140 Val Glu His Ile Ser Lys Gly Asn Asn Cys Leu Asp Ala Ala Lys Ala145 150 155 160 Cys Asn Leu Asp Asp Thr Cys Lys Lys Tyr Arg Ser Ala TyrIle Thr 165 170 175 Pro Cys Thr Thr Ser Met Ser Asn Glu Val Cys Asn ArgArg Lys Cys 180 185 190 His Lys Ala Leu Arg Gln Phe Phe Asp Lys Val ProAla Lys His Ser 195 200 205 Tyr Gly Met Leu Phe Cys Ser Cys Arg Asp IleAla Cys Thr Glu Arg 210 215 220 Arg Arg Gln Thr Ile Val Pro Val Cys SerTyr Glu Glu Arg Glu Arg 225 230 235 240 Pro Asn Cys Leu Ser Leu Gln AspSer Cys Lys Thr Asn Tyr Ile Cys 245 250 255 Arg Ser Arg Leu Ala Asp PhePhe Thr Asn Cys Gln Pro Glu Ser Arg 260 265 270 Ser Val Ser Asn Cys LeuLys Glu Asn Tyr Ala Asp Cys Leu Leu Ala 275 280 285 Tyr Ser Gly Leu IleGly Thr Val Met Thr Pro Asn Tyr Val Asp Ser 290 295 300 Ser Ser Leu SerVal Ala Pro Trp Cys Asp Cys Ser Asn Ser Gly Asn 305 310 315 320 Asp LeuGlu Asp Cys Leu Lys Phe Leu Asn Phe Phe Lys Asp Asn Thr 325 330 335 CysLeu Lys Asn Ala Ile Gln Ala Phe Gly Asn Gly Ser Asp Val Thr 340 345 350Met Trp Gln Pro Ala Pro Pro Val Gln Thr Thr Thr Ala Thr Thr Thr 355 360365 Thr Ala Phe Arg Val Lys Asn Lys Pro Leu Gly Pro Ala Gly Ser Glu 370375 380 Asn Glu Ile Pro Thr His Val Leu Pro Pro Cys Ala Asn Leu Gln Ala385 390 395 400 Gln Lys Leu Lys Ser Asn Val Ser Gly Ser Thr His Leu CysLeu Ser 405 410 415 Asp Ser Asp Phe Gly Lys Asp Gly Leu Ala Gly Ala SerSer His Ile 420 425 430 Thr Thr Lys Ser Met Ala Ala Pro Pro Ser Cys SerLeu Ser Ser Leu 435 440 445 Pro Val Leu Met Leu Thr Ala Leu Ala Ala LeuLeu Ser Val Ser Leu 450 455 460 Ala Glu Thr Ser 465 5 3209 DNA HUMAN CDS(540)..(1937) 5 aatctggcct cggaacacgc cattctccgc gccgcttcca ataaccactaacatccctaa 60 cgagcatccg agccgagggc tctgctcgga aatcgtcctg gcccaactcggcccttcgag 120 ctctcgaaga ttaccgcatc tatttttttt ttcttttttt tcttttcctagcgcagataa 180 agtgagcccg gaaagggaag gagggggcgg ggacaccatt gccctgaaagaataaataag 240 taaataaaca aactggctcc tcgccgcagc tggacgcggt cggttgagtccaggttgggt 300 cggacctgaa cccctaaaag cggaaccgcc tcccgccctc gccatcccggagctgagtcg 360 ccggcggcgg tggctgctgc cagacccgga gtttcctctt tcactggatggagctgaact 420 ttgggcggcc agagcagcac agctgtccgg ggatcgctgc acgctgagctccctcggcaa 480 gacccagcgg cggctcggga tttttttggg ggggcgggga ccagccccgcgccggcacc 539 atg ttc ctg gcg acc ctg tac ttc gcg ctg ccg ctc ttg gacttg ctc 587 Met Phe Leu Ala Thr Leu Tyr Phe Ala Leu Pro Leu Leu Asp LeuLeu 1 5 10 15 ctg tcg gcc gaa gtg agc ggc gga gac cgc ctg gat tgc gtgaaa gcc 635 Leu Ser Ala Glu Val Ser Gly Gly Asp Arg Leu Asp Cys Val LysAla 20 25 30 agt gat cag tgc ctg aag gag cag agc tgc agc acc aag tac cgcacg 683 Ser Asp Gln Cys Leu Lys Glu Gln Ser Cys Ser Thr Lys Tyr Arg Thr35 40 45 cta agg cag tgc gtg gcg ggc aag gag acc aac ttc agc ctg gca tcc731 Leu Arg Gln Cys Val Ala Gly Lys Glu Thr Asn Phe Ser Leu Ala Ser 5055 60 ggc ctg gag gcc aag gat gag tgc cgc agc gcc atg gag gcc ctg aag779 Gly Leu Glu Ala Lys Asp Glu Cys Arg Ser Ala Met Glu Ala Leu Lys 6570 75 80 cag aag tcg ctc tac aac tgc cgc tgc aag cgg ggt atg aag aag gag827 Gln Lys Ser Leu Tyr Asn Cys Arg Cys Lys Arg Gly Met Lys Lys Glu 8590 95 aag aac tgc ctg cgc att tac tgg agc atg tac cag agc ctg cag gga875 Lys Asn Cys Leu Arg Ile Tyr Trp Ser Met Tyr Gln Ser Leu Gln Gly 100105 110 aat gat ctg ctg gag gat tcc cca tat gaa cca gtt aac agc aga ttg923 Asn Asp Leu Leu Glu Asp Ser Pro Tyr Glu Pro Val Asn Ser Arg Leu 115120 125 tca gat ata ttc cgg gtg gtc cca ttc ata tca gat gtt ttt cag caa971 Ser Asp Ile Phe Arg Val Val Pro Phe Ile Ser Asp Val Phe Gln Gln 130135 140 gtg gag cac att ccc aaa ggg aac aac tgc ctg gat gca gcg aag gcc1019 Val Glu His Ile Pro Lys Gly Asn Asn Cys Leu Asp Ala Ala Lys Ala 145150 155 160 tgc aac ctc gac gac att tgc aag aag tac agg tcg gcg tac atcacc 1067 Cys Asn Leu Asp Asp Ile Cys Lys Lys Tyr Arg Ser Ala Tyr Ile Thr165 170 175 ccg tgc acc acc agc gtg tcc aan gat gtc tgc aac cgc cgc aagtgc 1115 Pro Cys Thr Thr Ser Val Ser Xaa Asp Val Cys Asn Arg Arg Lys Cys180 185 190 cac aag gcc ctc cgg cag ttc ttt gac aag gtc ccg gcc aag cacagc 1163 His Lys Ala Leu Arg Gln Phe Phe Asp Lys Val Pro Ala Lys His Ser195 200 205 tac gga atg ctc ttc tgc tcc tgc cgg gac atc gcc tgc aca gagcgg 1211 Tyr Gly Met Leu Phe Cys Ser Cys Arg Asp Ile Ala Cys Thr Glu Arg210 215 220 agg cga cag acc atc gtg cct gtg tgc tcc tat gaa gag agg gagaag 1259 Arg Arg Gln Thr Ile Val Pro Val Cys Ser Tyr Glu Glu Arg Glu Lys225 230 235 240 ccc aac tgt ttg aat ttg cag gac tcc tgc aag acg aat tacatc tgc 1307 Pro Asn Cys Leu Asn Leu Gln Asp Ser Cys Lys Thr Asn Tyr IleCys 245 250 255 aga tct cgc ctt gcg gat ttt ttt acc aac tgc cag cca gagtca agg 1355 Arg Ser Arg Leu Ala Asp Phe Phe Thr Asn Cys Gln Pro Glu SerArg 260 265 270 tct gtc agc agc tgt cta aag gaa aac tac gct gac tgc ctcctc gcc 1403 Ser Val Ser Ser Cys Leu Lys Glu Asn Tyr Ala Asp Cys Leu LeuAla 275 280 285 tac tcg ggg ctt att ggc aca gtc atg acc ccc aac tac atagac tcc 1451 Tyr Ser Gly Leu Ile Gly Thr Val Met Thr Pro Asn Tyr Ile AspSer 290 295 300 agt agc ctc agt gtg gcc cca tgg tgt gac tgc agc aac agtggg aac 1499 Ser Ser Leu Ser Val Ala Pro Trp Cys Asp Cys Ser Asn Ser GlyAsn 305 310 315 320 gac cta gaa gag tgc ttg aaa ttt ttg aat ttc ttc aaggac aat aca 1547 Asp Leu Glu Glu Cys Leu Lys Phe Leu Asn Phe Phe Lys AspAsn Thr 325 330 335 tgt ctt aaa aat gca att caa gcc ttt ggc aat ggc tccgat gtg acc 1595 Cys Leu Lys Asn Ala Ile Gln Ala Phe Gly Asn Gly Ser AspVal Thr 340 345 350 gtg tgg cag cca gcc ttc cca gta cag acc acc act gccact acc acc 1643 Val Trp Gln Pro Ala Phe Pro Val Gln Thr Thr Thr Ala ThrThr Thr 355 360 365 act gcc ctc cgg gtt aag aac aag ccc ctg ggg cca gcaggg tct gag 1691 Thr Ala Leu Arg Val Lys Asn Lys Pro Leu Gly Pro Ala GlySer Glu 370 375 380 aat gaa att ccc act cat gtt ttg cca ccg tgt gca aattta cag gca 1739 Asn Glu Ile Pro Thr His Val Leu Pro Pro Cys Ala Asn LeuGln Ala 385 390 395 400 cag aag ctg aaa tcc aat gtg tcg ggc aat aca cacctc tgt att tcc 1787 Gln Lys Leu Lys Ser Asn Val Ser Gly Asn Thr His LeuCys Ile Ser 405 410 415 aat ggt aat tat gaa aaa gaa ggt ctc ggt gct tccagc cac ata acc 1835 Asn Gly Asn Tyr Glu Lys Glu Gly Leu Gly Ala Ser SerHis Ile Thr 420 425 430 aca aaa tca atg gct gct cct cca agc tgt ggt ctgagc cca ctg ctg 1883 Thr Lys Ser Met Ala Ala Pro Pro Ser Cys Gly Leu SerPro Leu Leu 435 440 445 gtc ctg gtg gta acc gct ctg tcc acc cta tta tcttta aca gaa aca 1931 Val Leu Val Val Thr Ala Leu Ser Thr Leu Leu Ser LeuThr Glu Thr 450 455 460 tca tag ctgcattaaa aaaatacaat atggacatgtaaaaagacaa aaaccaagtt 1987 Ser 465 atctgtttcc tgttctcttg tatagctgaaattccagttt aggagctcag ttgagaaaca 2047 gttccattca actggaacat ttttttttttnccttttaag aaagcttctt gtgatccttc 2107 ggggcttctg tgaaaaacct gatgcagtgctccatccaaa ctcagaaggc tttgggatat 2167 gctgtatttt aaagggacag tttgtaacttgggctgtaaa gcaaactggg gctgtgtttt 2227 cgatgatgat gatcatcatg atcatgatnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2287 nnnnnnngat tttaacagtt ttacttctggcctttcctag ctagagaagg agttaatatt 2347 tctaaggtaa ctcccatatc tcctttaatgacattgattt ctaatgatat aaatttcagc 2407 ctacattgat gccaagcttt tttgccacaaagaagattct taccaagagt gggctttgtg 2467 gaaacagctg gtactgatgt tcacctttatatatgtacta gcattttcca cgctgatgtt 2527 tatgtactgt aaacagttct gcactcttgtacaaaagaaa aaacacctgt cacatccaaa 2587 tatagtatct gtcttttcgt caaaatagagagtggggaat gagtgtgccg attcaatacc 2647 tcaatccctg aacgacactc tcctaatcctaagccttacc tgagtgagaa gccctttacc 2707 taacaaaagt ccaatatagc tgaaatgtcgctctaatact ctttacacat atgaggttat 2767 atgtagaaaa aaattttact actaaatgatttcaactatt ggctttctat attttgaaag 2827 taatgatatt gtctcatttt tttactgatggtttaataca aaatacacag agcttgtttc 2887 ccctcataag tagtgttcgc tctgatatgaacttcacaaa tacagctcat caaaagcaga 2947 ctctgagaag cctcgtgctg tagcagaaagttctgcatca tgtgactgtg gacaggcagg 3007 aggaaacaga acagacaagc attgtcttttgtcattgctc gaagtgcaag cgtgcatacc 3067 tgtggaggga actggtggct gcttgtaaatgttctgcagc atctcttgac acacttgtca 3127 tgacacaatc cagtaccttg gttttcaggttatctgacaa aggcagcttt gattgggaca 3187 tggaggcatg ggcaggccgg aa 3209 6465 PRT HUMAN misc_feature (184)..(184) The ′Xaa′ at location 184 standsfor Lys, or Asn. 6 Met Phe Leu Ala Thr Leu Tyr Phe Ala Leu Pro Leu LeuAsp Leu Leu 1 5 10 15 Leu Ser Ala Glu Val Ser Gly Gly Asp Arg Leu AspCys Val Lys Ala 20 25 30 Ser Asp Gln Cys Leu Lys Glu Gln Ser Cys Ser ThrLys Tyr Arg Thr 35 40 45 Leu Arg Gln Cys Val Ala Gly Lys Glu Thr Asn PheSer Leu Ala Ser 50 55 60 Gly Leu Glu Ala Lys Asp Glu Cys Arg Ser Ala MetGlu Ala Leu Lys 65 70 75 80 Gln Lys Ser Leu Tyr Asn Cys Arg Cys Lys ArgGly Met Lys Lys Glu 85 90 95 Lys Asn Cys Leu Arg Ile Tyr Trp Ser Met TyrGln Ser Leu Gln Gly 100 105 110 Asn Asp Leu Leu Glu Asp Ser Pro Tyr GluPro Val Asn Ser Arg Leu 115 120 125 Ser Asp Ile Phe Arg Val Val Pro PheIle Ser Asp Val Phe Gln Gln 130 135 140 Val Glu His Ile Pro Lys Gly AsnAsn Cys Leu Asp Ala Ala Lys Ala 145 150 155 160 Cys Asn Leu Asp Asp IleCys Lys Lys Tyr Arg Ser Ala Tyr Ile Thr 165 170 175 Pro Cys Thr Thr SerVal Ser Xaa Asp Val Cys Asn Arg Arg Lys Cys 180 185 190 His Lys Ala LeuArg Gln Phe Phe Asp Lys Val Pro Ala Lys His Ser 195 200 205 Tyr Gly MetLeu Phe Cys Ser Cys Arg Asp Ile Ala Cys Thr Glu Arg 210 215 220 Arg ArgGln Thr Ile Val Pro Val Cys Ser Tyr Glu Glu Arg Glu Lys 225 230 235 240Pro Asn Cys Leu Asn Leu Gln Asp Ser Cys Lys Thr Asn Tyr Ile Cys 245 250255 Arg Ser Arg Leu Ala Asp Phe Phe Thr Asn Cys Gln Pro Glu Ser Arg 260265 270 Ser Val Ser Ser Cys Leu Lys Glu Asn Tyr Ala Asp Cys Leu Leu Ala275 280 285 Tyr Ser Gly Leu Ile Gly Thr Val Met Thr Pro Asn Tyr Ile AspSer 290 295 300 Ser Ser Leu Ser Val Ala Pro Trp Cys Asp Cys Ser Asn SerGly Asn 305 310 315 320 Asp Leu Glu Glu Cys Leu Lys Phe Leu Asn Phe PheLys Asp Asn Thr 325 330 335 Cys Leu Lys Asn Ala Ile Gln Ala Phe Gly AsnGly Ser Asp Val Thr 340 345 350 Val Trp Gln Pro Ala Phe Pro Val Gln ThrThr Thr Ala Thr Thr Thr 355 360 365 Thr Ala Leu Arg Val Lys Asn Lys ProLeu Gly Pro Ala Gly Ser Glu 370 375 380 Asn Glu Ile Pro Thr His Val LeuPro Pro Cys Ala Asn Leu Gln Ala 385 390 395 400 Gln Lys Leu Lys Ser AsnVal Ser Gly Asn Thr His Leu Cys Ile Ser 405 410 415 Asn Gly Asn Tyr GluLys Glu Gly Leu Gly Ala Ser Ser His Ile Thr 420 425 430 Thr Lys Ser MetAla Ala Pro Pro Ser Cys Gly Leu Ser Pro Leu Leu 435 440 445 Val Leu ValVal Thr Ala Leu Ser Thr Leu Leu Ser Leu Thr Glu Thr 450 455 460 Ser 4657 508 DNA HUMAN misc_feature (1)..(508) Note=“1 to 508 is -235 to 272 ofFigure 5 Hsgr- 21af” 7 tctggcctcg gaacacgcca ttctccgcgc cgcttccaataaccactaac atccctaacg 60 agcatccgag ccgagggctc tgctcggaaa tcgtcctggcccaactcggc ccttcgagct 120 ctcgaagatt accgcatcta tttttttttt cttttttttcttttcctagc gcagataaag 180 tgagcccgga aagggaagga gggggcgggg acaccattgccctgaaagaa taaataagta 240 aataaacaaa ctggctcctc gccgcagctg gacgcggtcggttgagtcca ggttgggtcg 300 gacctgaacc cctaaaagcg gaaccgcctc ccgccctcgccatcccggag ctgagtcgcc 360 ggcggcggtg gctgctgcca gacccggagt ttcctctttcactggatgga gctgaacttt 420 gggcggccag agcagcacag ctgtccgggg atcgctgcacgctgagctcc ctcggcaaga 480 cccagcggcg gctcgggatt tttttggg 508 8 510 DNAHUMAN misc_feature (1)..(510) Note=“1 to 510 is -237 to 272 of Figure 5Hsgr- 21bf” 8 aatctggcct cggaacacgc cattctccgc gccgcttcca ataaccactaacatccctaa 60 cgagcatccg agccgagggc tctgctcgga aatcgtcctg gcccaactcggcccttcgag 120 ctctcgaaga ttaccgcatc tatttttttt ttcttttttt tcttttcctagcgcagataa 180 agtgagcccg gaaagggaag gagggggcgg ggacaccatt gccctgaaagaataaataag 240 taaataaaca aactggctcc tcgccgcagc tggacgcggt cggttgagtccaggttgggt 300 cggacctgaa cccctaaaag cggaaccgcc tcccgccctc gccatcccggagctgagtcg 360 ccggcggcgg tggctgctgc cagacccgga gtttcctctt tcactggatggagctgaact 420 ttgggcggcc agagcagcac agctgtccgg ggatcgctgc acgctgagctccctcggcaa 480 gacccagcgg cggctcggga tttttttggg 510 9 1927 DNA HUMAN CDS(538)..(1926) 9 tctggcctcg gaacacgcca ttctccgcgc cgcttccaat aaccactaacatccctaacg 60 agcatccgag ccgagggctc tgctcggaaa tcgtcctggc ccaactcggcccttcgagct 120 ctcgaagatt accgcatcta tttttttttt cttttttttc ttttcctagcgcagataaag 180 tgagcccgga aagggaagga gggggcgggg acaccattgc cctgaaagaataaataagta 240 aataaacaaa ctggctcctc gccgcagctg gacgcggtcg gttgagtccaggttgggtcg 300 gacctgaacc cctaaaagcg gaaccgcctc ccgccctcgc catcccggagctgagtcgcc 360 ggcggcggtg gctgctgcca gacccggagt ttcctctttc actggatggagctgaacttt 420 gggcggccag agcagcacag ctgtccgggg atcgctgcac gctgagctccctcggcaaga 480 cccagcggcg gctcgggatt tttttggggg ggcggggacc agccccgcgccggcacc 537 atg ttc ctg gcg ncc ctg tac ttc gcg ctg ccg ctc ttg gac ttgctc 585 Met Phe Leu Ala Xaa Leu Tyr Phe Ala Leu Pro Leu Leu Asp Leu Leu1 5 10 15 ctg tcg gcc gaa gtg agc ggc gga gac cgc ctg gat tgc gtg aaagcc 633 Leu Ser Ala Glu Val Ser Gly Gly Asp Arg Leu Asp Cys Val Lys Ala20 25 30 agt gat cag tgc ctg aag gag cag agc tgc agc acc aag tac cgc acg681 Ser Asp Gln Cys Leu Lys Glu Gln Ser Cys Ser Thr Lys Tyr Arg Thr 3540 45 cta agg cag tgc gtg gcg ggc aag gag acc aac ttc agc ctg gca tcc729 Leu Arg Gln Cys Val Ala Gly Lys Glu Thr Asn Phe Ser Leu Ala Ser 5055 60 ggc ctg gag gcc aag gat gag tgc cgc agc gcc atg gag gcc ctg aag777 Gly Leu Glu Ala Lys Asp Glu Cys Arg Ser Ala Met Glu Ala Leu Lys 6570 75 80 cag aag tcg ctc tac aac tgc cgc tgc aag cgg ggt atg aag aag gag825 Gln Lys Ser Leu Tyr Asn Cys Arg Cys Lys Arg Gly Met Lys Lys Glu 8590 95 aag aac tgc ctg cgc att tac tgg agc atg tac cag agc ctg cag gga873 Lys Asn Cys Leu Arg Ile Tyr Trp Ser Met Tyr Gln Ser Leu Gln Gly 100105 110 aat gat ctg ctg gag gat tcc cca tat gaa cca gtt aac agc aga ttg921 Asn Asp Leu Leu Glu Asp Ser Pro Tyr Glu Pro Val Asn Ser Arg Leu 115120 125 tca gat ata ttc cgg gtg gtc cca ttc ata tca gat gtt ttt cag caa969 Ser Asp Ile Phe Arg Val Val Pro Phe Ile Ser Asp Val Phe Gln Gln 130135 140 gtg gag cac att ccc aaa ggg aac aac tgc ctg gat gca gcg aag gcc1017 Val Glu His Ile Pro Lys Gly Asn Asn Cys Leu Asp Ala Ala Lys Ala 145150 155 160 tgc aac ctc gac gac att tgc aag aag tac agg tcg gcg tac atcacc 1065 Cys Asn Leu Asp Asp Ile Cys Lys Lys Tyr Arg Ser Ala Tyr Ile Thr165 170 175 ccg tgc acc acc agc gtg tcc aac gat gtc tgc aac cgc cgc aagtgc 1113 Pro Cys Thr Thr Ser Val Ser Asn Asp Val Cys Asn Arg Arg Lys Cys180 185 190 cac aag gcc ctc cgg cag ttc ttt gac aag gtc ccg gcc aag cacagc 1161 His Lys Ala Leu Arg Gln Phe Phe Asp Lys Val Pro Ala Lys His Ser195 200 205 tac gga atg ctc ttc tgc tcc tgc cgg gac atc gcc tgc aca gagcgg 1209 Tyr Gly Met Leu Phe Cys Ser Cys Arg Asp Ile Ala Cys Thr Glu Arg210 215 220 agg cga cag acc atc gtg cct gtg tgc tcc tat gaa gag agg gagaag 1257 Arg Arg Gln Thr Ile Val Pro Val Cys Ser Tyr Glu Glu Arg Glu Lys225 230 235 240 ccc aac tgt ttg aat ttg cag gac tcc tgc aag acg aat tacatc tgc 1305 Pro Asn Cys Leu Asn Leu Gln Asp Ser Cys Lys Thr Asn Tyr IleCys 245 250 255 aga tct cgc ctt gcg gat ttt ttt acc aac tgc cag cca gagtca agg 1353 Arg Ser Arg Leu Ala Asp Phe Phe Thr Asn Cys Gln Pro Glu SerArg 260 265 270 tct gtc agc agc tgt cta aag gaa aac tac gct gac tgc ctcctc gcc 1401 Ser Val Ser Ser Cys Leu Lys Glu Asn Tyr Ala Asp Cys Leu LeuAla 275 280 285 tac tcg ggg ctt att ggc aca gtc atg acc ccc aac tac atagac tcc 1449 Tyr Ser Gly Leu Ile Gly Thr Val Met Thr Pro Asn Tyr Ile AspSer 290 295 300 agt agc ctc agt gtg gcc cca tgg tgt gac tgc agc aac agtggg aac 1497 Ser Ser Leu Ser Val Ala Pro Trp Cys Asp Cys Ser Asn Ser GlyAsn 305 310 315 320 gac cta gaa gag tgc ttg aaa ttt ttg aat ttc ttc aaggac aat aca 1545 Asp Leu Glu Glu Cys Leu Lys Phe Leu Asn Phe Phe Lys AspAsn Thr 325 330 335 tgt ctt aaa aat gca att caa gcc ttt ggc aat ggc tccgat gtg acc 1593 Cys Leu Lys Asn Ala Ile Gln Ala Phe Gly Asn Gly Ser AspVal Thr 340 345 350 gtg tgg cag cca gcc ttc cca gta cag acc acc act gccact acc acc 1641 Val Trp Gln Pro Ala Phe Pro Val Gln Thr Thr Thr Ala ThrThr Thr 355 360 365 act gcc ctc cgg gtt aag aac aag ccc ctg ggg cca gcaggg tct gag 1689 Thr Ala Leu Arg Val Lys Asn Lys Pro Leu Gly Pro Ala GlySer Glu 370 375 380 aat gaa att ccc act cat gtt ttg cca ccg tgt gca aattta cag gca 1737 Asn Glu Ile Pro Thr His Val Leu Pro Pro Cys Ala Asn LeuGln Ala 385 390 395 400 cag aag ctg aaa tcc aat gtg tcg ggc aat aca cacctc tgt att tcc 1785 Gln Lys Leu Lys Ser Asn Val Ser Gly Asn Thr His LeuCys Ile Ser 405 410 415 aat ggt aat tat gaa aaa gaa ggt ctc ggt gct tccagc cac ata acc 1833 Asn Gly Asn Tyr Glu Lys Glu Gly Leu Gly Ala Ser SerHis Ile Thr 420 425 430 aca aaa tca atg gct gct cct cca agc tgt ggt ctgagc cca ctg ctg 1881 Thr Lys Ser Met Ala Ala Pro Pro Ser Cys Gly Leu SerPro Leu Leu 435 440 445 gtc ctg gtg gta acc gct ctg tcc acc cta tta tcttta aca gaa a 1927 Val Leu Val Val Thr Ala Leu Ser Thr Leu Leu Ser LeuThr Glu 450 455 460 10 463 PRT HUMAN misc_feature (5)..(5) The ′Xaa′ atlocation 5 stands for Thr, Ala, Pro, or Ser. 10 Met Phe Leu Ala Xaa LeuTyr Phe Ala Leu Pro Leu Leu Asp Leu Leu 1 5 10 15 Leu Ser Ala Glu ValSer Gly Gly Asp Arg Leu Asp Cys Val Lys Ala 20 25 30 Ser Asp Gln Cys LeuLys Glu Gln Ser Cys Ser Thr Lys Tyr Arg Thr 35 40 45 Leu Arg Gln Cys ValAla Gly Lys Glu Thr Asn Phe Ser Leu Ala Ser 50 55 60 Gly Leu Glu Ala LysAsp Glu Cys Arg Ser Ala Met Glu Ala Leu Lys 65 70 75 80 Gln Lys Ser LeuTyr Asn Cys Arg Cys Lys Arg Gly Met Lys Lys Glu 85 90 95 Lys Asn Cys LeuArg Ile Tyr Trp Ser Met Tyr Gln Ser Leu Gln Gly 100 105 110 Asn Asp LeuLeu Glu Asp Ser Pro Tyr Glu Pro Val Asn Ser Arg Leu 115 120 125 Ser AspIle Phe Arg Val Val Pro Phe Ile Ser Asp Val Phe Gln Gln 130 135 140 ValGlu His Ile Pro Lys Gly Asn Asn Cys Leu Asp Ala Ala Lys Ala 145 150 155160 Cys Asn Leu Asp Asp Ile Cys Lys Lys Tyr Arg Ser Ala Tyr Ile Thr 165170 175 Pro Cys Thr Thr Ser Val Ser Asn Asp Val Cys Asn Arg Arg Lys Cys180 185 190 His Lys Ala Leu Arg Gln Phe Phe Asp Lys Val Pro Ala Lys HisSer 195 200 205 Tyr Gly Met Leu Phe Cys Ser Cys Arg Asp Ile Ala Cys ThrGlu Arg 210 215 220 Arg Arg Gln Thr Ile Val Pro Val Cys Ser Tyr Glu GluArg Glu Lys 225 230 235 240 Pro Asn Cys Leu Asn Leu Gln Asp Ser Cys LysThr Asn Tyr Ile Cys 245 250 255 Arg Ser Arg Leu Ala Asp Phe Phe Thr AsnCys Gln Pro Glu Ser Arg 260 265 270 Ser Val Ser Ser Cys Leu Lys Glu AsnTyr Ala Asp Cys Leu Leu Ala 275 280 285 Tyr Ser Gly Leu Ile Gly Thr ValMet Thr Pro Asn Tyr Ile Asp Ser 290 295 300 Ser Ser Leu Ser Val Ala ProTrp Cys Asp Cys Ser Asn Ser Gly Asn 305 310 315 320 Asp Leu Glu Glu CysLeu Lys Phe Leu Asn Phe Phe Lys Asp Asn Thr 325 330 335 Cys Leu Lys AsnAla Ile Gln Ala Phe Gly Asn Gly Ser Asp Val Thr 340 345 350 Val Trp GlnPro Ala Phe Pro Val Gln Thr Thr Thr Ala Thr Thr Thr 355 360 365 Thr AlaLeu Arg Val Lys Asn Lys Pro Leu Gly Pro Ala Gly Ser Glu 370 375 380 AsnGlu Ile Pro Thr His Val Leu Pro Pro Cys Ala Asn Leu Gln Ala 385 390 395400 Gln Lys Leu Lys Ser Asn Val Ser Gly Asn Thr His Leu Cys Ile Ser 405410 415 Asn Gly Asn Tyr Glu Lys Glu Gly Leu Gly Ala Ser Ser His Ile Thr420 425 430 Thr Lys Ser Met Ala Ala Pro Pro Ser Cys Gly Leu Ser Pro LeuLeu 435 440 445 Val Leu Val Val Thr Ala Leu Ser Thr Leu Leu Ser Leu ThrGlu 450 455 460 11 1929 DNA HUMAN CDS (540)..(1928) 11 aatctggcctcggaacacgc cattctccgc gccgcttcca ataaccacta acatccctaa 60 cgagcatccgagccgagggc tctgctcgga aatcgtcctg gcccaactcg gcccttcgag 120 ctctcgaagattaccgcatc tatttttttt ttcttttttt tcttttccta gcgcagataa 180 agtgagcccggaaagggaag gagggggcgg ggacaccatt gccctgaaag aataaataag 240 taaataaacaaactggctcc tcgccgcagc tggacgcggt cggttgagtc caggttgggt 300 cggacctgaacccctaaaag cggaaccgcc tcccgccctc gccatcccgg agctgagtcg 360 ccggcggcggtggctgctgc cagacccgga gtttcctctt tcactggatg gagctgaact 420 ttgggcggccagagcagcac agctgtccgg ggatcgctgc acgctgagct ccctcggcaa 480 gacccagcggcggctcggga tttttttggg ggggcgggga ccagccccgc gccggcacc 539 atg ttc ctggcg acc ctg tac ttc gcg ctg ccg ctc ttg gac ttg ctc 587 Met Phe Leu AlaThr Leu Tyr Phe Ala Leu Pro Leu Leu Asp Leu Leu 1 5 10 15 ctg tcg gccgaa gtg agc ggc gga gac cgc ctg gat tgc gtg aaa gcc 635 Leu Ser Ala GluVal Ser Gly Gly Asp Arg Leu Asp Cys Val Lys Ala 20 25 30 agt gat cag tgcctg aag gag cag agc tgc agc acc aag tac cgc acg 683 Ser Asp Gln Cys LeuLys Glu Gln Ser Cys Ser Thr Lys Tyr Arg Thr 35 40 45 cta agg cag tgc gtggcg ggc aag gag acc aac ttc agc ctg gca tcc 731 Leu Arg Gln Cys Val AlaGly Lys Glu Thr Asn Phe Ser Leu Ala Ser 50 55 60 ggc ctg gag gcc aag gatgag tgc cgc agc gcc atg gag gcc ctg aag 779 Gly Leu Glu Ala Lys Asp GluCys Arg Ser Ala Met Glu Ala Leu Lys 65 70 75 80 cag aag tcg ctc tac aactgc cgc tgc aag cgg ggt atg aag aag gag 827 Gln Lys Ser Leu Tyr Asn CysArg Cys Lys Arg Gly Met Lys Lys Glu 85 90 95 aag aac tgc ctg cgc att tactgg agc atg tac cag agc ctg cag gga 875 Lys Asn Cys Leu Arg Ile Tyr TrpSer Met Tyr Gln Ser Leu Gln Gly 100 105 110 aat gat ctg ctg gag gat tcccca tat gaa cca gtt aac agc aga ttg 923 Asn Asp Leu Leu Glu Asp Ser ProTyr Glu Pro Val Asn Ser Arg Leu 115 120 125 tca gat ata ttc cgg gtg gtccca ttc ata tca gat gtt ttt cag caa 971 Ser Asp Ile Phe Arg Val Val ProPhe Ile Ser Asp Val Phe Gln Gln 130 135 140 gtg gag cac att ccc aaa gggaac aac tgc ctg gat gca gcg aag gcc 1019 Val Glu His Ile Pro Lys Gly AsnAsn Cys Leu Asp Ala Ala Lys Ala 145 150 155 160 tgc aac ctc gac gac atttgc aag aag tac agg tcg gcg tac atc acc 1067 Cys Asn Leu Asp Asp Ile CysLys Lys Tyr Arg Ser Ala Tyr Ile Thr 165 170 175 ccg tgc acc acc agc gtgtcc aac gat gtc tgc aac cgc cgc aag tgc 1115 Pro Cys Thr Thr Ser Val SerAsn Asp Val Cys Asn Arg Arg Lys Cys 180 185 190 cac aag gcc ctc cgg cagttc ttt gac aag gtc ccg gcc aag cac agc 1163 His Lys Ala Leu Arg Gln PhePhe Asp Lys Val Pro Ala Lys His Ser 195 200 205 tac gga atg ctc ttc tgctcc tgc cgg gac atc gcc tgc aca gag cgg 1211 Tyr Gly Met Leu Phe Cys SerCys Arg Asp Ile Ala Cys Thr Glu Arg 210 215 220 agg cga cag acc atc gtgcct gtg tgc tcc tat gaa gag agg gag aag 1259 Arg Arg Gln Thr Ile Val ProVal Cys Ser Tyr Glu Glu Arg Glu Lys 225 230 235 240 ccc aac tgt ttg aatttg cag gac tcc tgc aag acg aat tac atc tgc 1307 Pro Asn Cys Leu Asn LeuGln Asp Ser Cys Lys Thr Asn Tyr Ile Cys 245 250 255 aga tct cgc ctt gcggat ttt ttt acc aac tgc cag cca gag tca agg 1355 Arg Ser Arg Leu Ala AspPhe Phe Thr Asn Cys Gln Pro Glu Ser Arg 260 265 270 tct gtc agc agc tgtcta aag gaa aac tac gct gac tgc ctc ctc gcc 1403 Ser Val Ser Ser Cys LeuLys Glu Asn Tyr Ala Asp Cys Leu Leu Ala 275 280 285 tac tcg ggg ctt attggc aca gtc atg acc ccc aac tac ata gac tcc 1451 Tyr Ser Gly Leu Ile GlyThr Val Met Thr Pro Asn Tyr Ile Asp Ser 290 295 300 agt agc ctc agt gtggcc cca tgg tgt gac tgc agc aac agt ggg aac 1499 Ser Ser Leu Ser Val AlaPro Trp Cys Asp Cys Ser Asn Ser Gly Asn 305 310 315 320 gac cta gaa gagtgc ttg aaa ttt ttg aat ttc ttc aag gac aat aca 1547 Asp Leu Glu Glu CysLeu Lys Phe Leu Asn Phe Phe Lys Asp Asn Thr 325 330 335 tgt ctt aaa aatgca att caa gcc ttt ggc aat ggc tcc gat gtg acc 1595 Cys Leu Lys Asn AlaIle Gln Ala Phe Gly Asn Gly Ser Asp Val Thr 340 345 350 gtg tgg cag ccagcc ttc cca gta cag acc acc act gcc act acc acc 1643 Val Trp Gln Pro AlaPhe Pro Val Gln Thr Thr Thr Ala Thr Thr Thr 355 360 365 act gcc ctc cgggtt aag aac aag ccc ctg ggg cca gca ggg tct gag 1691 Thr Ala Leu Arg ValLys Asn Lys Pro Leu Gly Pro Ala Gly Ser Glu 370 375 380 aat gaa att cccact cat gtt ttg cca ccg tgt gca aat tta cag gca 1739 Asn Glu Ile Pro ThrHis Val Leu Pro Pro Cys Ala Asn Leu Gln Ala 385 390 395 400 cag aag ctgaaa tcc aat gtg tcg ggc aat aca cac ctc tgt att tcc 1787 Gln Lys Leu LysSer Asn Val Ser Gly Asn Thr His Leu Cys Ile Ser 405 410 415 aat ggt aattat gaa aaa gaa ggt ctc ggt gct tcc agc cac ata acc 1835 Asn Gly Asn TyrGlu Lys Glu Gly Leu Gly Ala Ser Ser His Ile Thr 420 425 430 aca aaa tcaatg gct gct cct cca agc tgt ggt ctg agc cca ctg ctg 1883 Thr Lys Ser MetAla Ala Pro Pro Ser Cys Gly Leu Ser Pro Leu Leu 435 440 445 gtc ctg gtggta acc gct ctg tcc acc cta tta tct tta aca gaa a 1929 Val Leu Val ValThr Ala Leu Ser Thr Leu Leu Ser Leu Thr Glu 450 455 460 12 463 PRT HUMANmisc_feature (1)..(539) Note= “1 to 539 is -237 to 301 of Figure 521bcon” 12 Met Phe Leu Ala Thr Leu Tyr Phe Ala Leu Pro Leu Leu Asp LeuLeu 1 5 10 15 Leu Ser Ala Glu Val Ser Gly Gly Asp Arg Leu Asp Cys ValLys Ala 20 25 30 Ser Asp Gln Cys Leu Lys Glu Gln Ser Cys Ser Thr Lys TyrArg Thr 35 40 45 Leu Arg Gln Cys Val Ala Gly Lys Glu Thr Asn Phe Ser LeuAla Ser 50 55 60 Gly Leu Glu Ala Lys Asp Glu Cys Arg Ser Ala Met Glu AlaLeu Lys 65 70 75 80 Gln Lys Ser Leu Tyr Asn Cys Arg Cys Lys Arg Gly MetLys Lys Glu 85 90 95 Lys Asn Cys Leu Arg Ile Tyr Trp Ser Met Tyr Gln SerLeu Gln Gly 100 105 110 Asn Asp Leu Leu Glu Asp Ser Pro Tyr Glu Pro ValAsn Ser Arg Leu 115 120 125 Ser Asp Ile Phe Arg Val Val Pro Phe Ile SerAsp Val Phe Gln Gln 130 135 140 Val Glu His Ile Pro Lys Gly Asn Asn CysLeu Asp Ala Ala Lys Ala 145 150 155 160 Cys Asn Leu Asp Asp Ile Cys LysLys Tyr Arg Ser Ala Tyr Ile Thr 165 170 175 Pro Cys Thr Thr Ser Val SerAsn Asp Val Cys Asn Arg Arg Lys Cys 180 185 190 His Lys Ala Leu Arg GlnPhe Phe Asp Lys Val Pro Ala Lys His Ser 195 200 205 Tyr Gly Met Leu PheCys Ser Cys Arg Asp Ile Ala Cys Thr Glu Arg 210 215 220 Arg Arg Gln ThrIle Val Pro Val Cys Ser Tyr Glu Glu Arg Glu Lys 225 230 235 240 Pro AsnCys Leu Asn Leu Gln Asp Ser Cys Lys Thr Asn Tyr Ile Cys 245 250 255 ArgSer Arg Leu Ala Asp Phe Phe Thr Asn Cys Gln Pro Glu Ser Arg 260 265 270Ser Val Ser Ser Cys Leu Lys Glu Asn Tyr Ala Asp Cys Leu Leu Ala 275 280285 Tyr Ser Gly Leu Ile Gly Thr Val Met Thr Pro Asn Tyr Ile Asp Ser 290295 300 Ser Ser Leu Ser Val Ala Pro Trp Cys Asp Cys Ser Asn Ser Gly Asn305 310 315 320 Asp Leu Glu Glu Cys Leu Lys Phe Leu Asn Phe Phe Lys AspAsn Thr 325 330 335 Cys Leu Lys Asn Ala Ile Gln Ala Phe Gly Asn Gly SerAsp Val Thr 340 345 350 Val Trp Gln Pro Ala Phe Pro Val Gln Thr Thr ThrAla Thr Thr Thr 355 360 365 Thr Ala Leu Arg Val Lys Asn Lys Pro Leu GlyPro Ala Gly Ser Glu 370 375 380 Asn Glu Ile Pro Thr His Val Leu Pro ProCys Ala Asn Leu Gln Ala 385 390 395 400 Gln Lys Leu Lys Ser Asn Val SerGly Asn Thr His Leu Cys Ile Ser 405 410 415 Asn Gly Asn Tyr Glu Lys GluGly Leu Gly Ala Ser Ser His Ile Thr 420 425 430 Thr Lys Ser Met Ala AlaPro Pro Ser Cys Gly Leu Ser Pro Leu Leu 435 440 445 Val Leu Val Val ThrAla Leu Ser Thr Leu Leu Ser Leu Thr Glu 450 455 460 13 699 DNA HUMAN CDS(2)..(697) 13 g tcg gcg tac atc acc ccg tgc acc acc agc gtg tcc aat gatgtc tgc 49 Ser Ala Tyr Ile Thr Pro Cys Thr Thr Ser Val Ser Asn Asp ValCys 1 5 10 15 aac cgc cgc aag tgc cac aag gcc ctc cgg cag ttc ttt gacaag gtc 97 Asn Arg Arg Lys Cys His Lys Ala Leu Arg Gln Phe Phe Asp LysVal 20 25 30 ccg gcc aag cac agc tac gga atg ctc ttc tgc tcc tgc cgg gacatc 145 Pro Ala Lys His Ser Tyr Gly Met Leu Phe Cys Ser Cys Arg Asp Ile35 40 45 gcc tgc aca gag cgg agg cga cag acc atc gtg cct gtg tgc tcc tat193 Ala Cys Thr Glu Arg Arg Arg Gln Thr Ile Val Pro Val Cys Ser Tyr 5055 60 gaa gag agg gag aag ccc aac tgt ttg aat ttg cag gac tcc tgc aag241 Glu Glu Arg Glu Lys Pro Asn Cys Leu Asn Leu Gln Asp Ser Cys Lys 6570 75 80 acg aat tac atc tgc aga tct cgc ctt gcg gat ttt ttt acc aac tgc289 Thr Asn Tyr Ile Cys Arg Ser Arg Leu Ala Asp Phe Phe Thr Asn Cys 8590 95 cag cca gag tca agg tct gtc agc agc tgt cta aag gaa aac tac gct337 Gln Pro Glu Ser Arg Ser Val Ser Ser Cys Leu Lys Glu Asn Tyr Ala 100105 110 gac tgc ctc ctc gcc tac tcg ggg ctt att ggc aca gtc atg acc ccc385 Asp Cys Leu Leu Ala Tyr Ser Gly Leu Ile Gly Thr Val Met Thr Pro 115120 125 aac tac ata gac tcc agt agc ctc agt gtg gcc cca tgg tgt gac tgc433 Asn Tyr Ile Asp Ser Ser Ser Leu Ser Val Ala Pro Trp Cys Asp Cys 130135 140 agc aac agt ggg aac gac cta gaa gag tgc ttg aaa ttt ttg aat ttc481 Ser Asn Ser Gly Asn Asp Leu Glu Glu Cys Leu Lys Phe Leu Asn Phe 145150 155 160 ttc aag gac aat aca tgt ctt aaa aat gca att caa gcc ttt ggcaat 529 Phe Lys Asp Asn Thr Cys Leu Lys Asn Ala Ile Gln Ala Phe Gly Asn165 170 175 ggc tcc gat gtg acc gtg tgg cag cca gcc ttc cca gta cag accacc 577 Gly Ser Asp Val Thr Val Trp Gln Pro Ala Phe Pro Val Gln Thr Thr180 185 190 act gcc gct acc acc act gcc ctc cgg gtt aag aac aag ccc ctgggg 625 Thr Ala Ala Thr Thr Thr Ala Leu Arg Val Lys Asn Lys Pro Leu Gly195 200 205 cca gca ggg tct gag aat gaa att ccc act cat gtt ttg cca ccgtgt 673 Pro Ala Gly Ser Glu Asn Glu Ile Pro Thr His Val Leu Pro Pro Cys210 215 220 gca aat tta cag gca cag aag ctg aa 699 Ala Asn Leu Gln AlaGln Lys Leu 225 230 14 232 PRT HUMAN misc_feature (1)..(699) Note= “1 to699 is 814 to 1512 of Figure 5 Hsgr-29a” 14 Ser Ala Tyr Ile Thr Pro CysThr Thr Ser Val Ser Asn Asp Val Cys 1 5 10 15 Asn Arg Arg Lys Cys HisLys Ala Leu Arg Gln Phe Phe Asp Lys Val 20 25 30 Pro Ala Lys His Ser TyrGly Met Leu Phe Cys Ser Cys Arg Asp Ile 35 40 45 Ala Cys Thr Glu Arg ArgArg Gln Thr Ile Val Pro Val Cys Ser Tyr 50 55 60 Glu Glu Arg Glu Lys ProAsn Cys Leu Asn Leu Gln Asp Ser Cys Lys 65 70 75 80 Thr Asn Tyr Ile CysArg Ser Arg Leu Ala Asp Phe Phe Thr Asn Cys 85 90 95 Gln Pro Glu Ser ArgSer Val Ser Ser Cys Leu Lys Glu Asn Tyr Ala 100 105 110 Asp Cys Leu LeuAla Tyr Ser Gly Leu Ile Gly Thr Val Met Thr Pro 115 120 125 Asn Tyr IleAsp Ser Ser Ser Leu Ser Val Ala Pro Trp Cys Asp Cys 130 135 140 Ser AsnSer Gly Asn Asp Leu Glu Glu Cys Leu Lys Phe Leu Asn Phe 145 150 155 160Phe Lys Asp Asn Thr Cys Leu Lys Asn Ala Ile Gln Ala Phe Gly Asn 165 170175 Gly Ser Asp Val Thr Val Trp Gln Pro Ala Phe Pro Val Gln Thr Thr 180185 190 Thr Ala Ala Thr Thr Thr Ala Leu Arg Val Lys Asn Lys Pro Leu Gly195 200 205 Pro Ala Gly Ser Glu Asn Glu Ile Pro Thr His Val Leu Pro ProCys 210 215 220 Ala Asn Leu Gln Ala Gln Lys Leu 225 230 15 2157 DNAHUMAN CDS (2)..(886) 15 g tcg gcg tac atc acc ccg tgc acc acc agc gtgtcc aat gat gtc tgc 49 Ser Ala Tyr Ile Thr Pro Cys Thr Thr Ser Val SerAsn Asp Val Cys 1 5 10 15 aac cgc cgc aag tgc cac aag gcc ctc cgg cagttc ttt gac aag gtc 97 Asn Arg Arg Lys Cys His Lys Ala Leu Arg Gln PhePhe Asp Lys Val 20 25 30 ccg gcc aag cac agc tac gga atg ctc ttc tgc tcctgc cgg gac atc 145 Pro Ala Lys His Ser Tyr Gly Met Leu Phe Cys Ser CysArg Asp Ile 35 40 45 gcc tgc aca gag cgg agg cga cag acc atc gtg cct gtgtgc tcc tat 193 Ala Cys Thr Glu Arg Arg Arg Gln Thr Ile Val Pro Val CysSer Tyr 50 55 60 gaa gag agg gag aag ccc aac tgt ttg aat ttg cag gac tcctgc aag 241 Glu Glu Arg Glu Lys Pro Asn Cys Leu Asn Leu Gln Asp Ser CysLys 65 70 75 80 acg aat tac atc tgc aga tct cgc ctt gcg gat ttt ttt accaac tgc 289 Thr Asn Tyr Ile Cys Arg Ser Arg Leu Ala Asp Phe Phe Thr AsnCys 85 90 95 cag cca gag tca agg tct gtc agc agc tgt cta aag gaa aac tacgct 337 Gln Pro Glu Ser Arg Ser Val Ser Ser Cys Leu Lys Glu Asn Tyr Ala100 105 110 gac tgc ctc ctc gcc tac tcg ggg ctt att ggc aca gtc atg accccc 385 Asp Cys Leu Leu Ala Tyr Ser Gly Leu Ile Gly Thr Val Met Thr Pro115 120 125 aac tac ata gac tcc agt agc ctc agt gtg gcc cca tgg tgt gactgc 433 Asn Tyr Ile Asp Ser Ser Ser Leu Ser Val Ala Pro Trp Cys Asp Cys130 135 140 agc aac agt ggg aac gac cta gaa gag tgc ttg aaa ttt ttg aatttc 481 Ser Asn Ser Gly Asn Asp Leu Glu Glu Cys Leu Lys Phe Leu Asn Phe145 150 155 160 ttc aag gac aat aca tgt ctt aaa aat gca att caa gcc tttggc aat 529 Phe Lys Asp Asn Thr Cys Leu Lys Asn Ala Ile Gln Ala Phe GlyAsn 165 170 175 ggc tcc gat gtg acc gtg tgg cag cca gcc ttc cca gta cagacc acc 577 Gly Ser Asp Val Thr Val Trp Gln Pro Ala Phe Pro Val Gln ThrThr 180 185 190 act gcc gct acc acc act gcc ctc cgg gtt aag aac aag cccctg ggg 625 Thr Ala Ala Thr Thr Thr Ala Leu Arg Val Lys Asn Lys Pro LeuGly 195 200 205 cca gca ggg tct gag aat gaa att ccc act cat gtt ttg ccaccg tgt 673 Pro Ala Gly Ser Glu Asn Glu Ile Pro Thr His Val Leu Pro ProCys 210 215 220 gca aat tta cag gca cag aag ctg aaa tcc aat gtg tcg ggcaat aca 721 Ala Asn Leu Gln Ala Gln Lys Leu Lys Ser Asn Val Ser Gly AsnThr 225 230 235 240 cac ctc tgt att tcc aat ggt aat tat gaa aaa gaa ggtctc ggt gct 769 His Leu Cys Ile Ser Asn Gly Asn Tyr Glu Lys Glu Gly LeuGly Ala 245 250 255 tcc agc cac ata acc aca aaa tca atg gct gct cct ccaagc tgt ggt 817 Ser Ser His Ile Thr Thr Lys Ser Met Ala Ala Pro Pro SerCys Gly 260 265 270 ctg agc cca ctg ctg gtc ctg gtg gta acc gct ctg tccacc cta tta 865 Leu Ser Pro Leu Leu Val Leu Val Val Thr Ala Leu Ser ThrLeu Leu 275 280 285 tct tta aca gaa aca tca tag ctgcattaaa aaaatacaatatggacatgt 916 Ser Leu Thr Glu Thr Ser 290 aaaaagacaa aaaccaagttatctgtttcc tgttctcttg tatagctgaa attccagttt 976 aggagctcag ttgagaaacagttccattca actggaacat tttttttttt ccttttaaga 1036 aagcttcttg tgatccttcggggcttctgt gaaaaacctg atgcagtgct ccatccaaac 1096 tcagaaggct ttgggatatgctgtatttta aagggacagt ttgtaacttg ggctgtaaag 1156 caaactgggg ctgtgttttcgatgatgatg atcatcatga tcatgatnnn nnnnnnnnnn 1216 nnnnnnnnnn nnnnnnnnnnnnnnnngatt ttaacagttt tacttctggc ctttcctagc 1276 tagagaagga gttaatatttctaaggtaac tcccatatct cctttaatga cattgatttc 1336 taatgatata aatttcagcctacattgatg ccaagctttt ttgccacaaa gaagattctt 1396 accaagagtg ggctttgtggaaacagctgg tactgatgtt cacctttata tatgtactag 1456 cattttccac gctgatgtttatgtactgta aacagttctg cactcttgta caaaagaaaa 1516 aacacctgtc acatccaaatatagtatctg tcttttcgtc aaaatagaga gtggggaatg 1576 agtgtgccga ttcaatacctcaatccctga acgacactct cctaatccta agccttacct 1636 gagtgagaag ccctttacctaacaaaagtc caatatagct gaaatgtcgc tctaatactc 1696 tttacacata tgaggttatatgtagaaaaa aattttacta ctaaatgatt tcaactattg 1756 gctttctata ttttgaaagtaatgatattg tctcattttt ttactgatgg tttaatacaa 1816 aatacacaga gcttgtttcccctcataagt agtgttcgct ctgatatgaa cttcacaaat 1876 acagctcatc aaaagcagactctgagaagc ctcgtgctgt agcagaaagt tctgcatcat 1936 gtgactgtgg acaggcaggaggaaacagaa cagacaagca ttgtcttttg tcattgctcg 1996 aagtgcaagc gtgcatacctgtggagggaa ctggtggctg cttgtaaatg ttctgcagca 2056 tctcttgaca cacttgtcatgacacaatcc agtaccttgg ttttcaggtt atctgacaaa 2116 ggcagctttg attgggacatggaggcatgg gcaggccgga a 2157 16 294 PRT HUMAN misc_feature (1)..(2157)Note= “1 to 2157 is 814 to 2971 of Figure 5 29brc” 16 Ser Ala Tyr IleThr Pro Cys Thr Thr Ser Val Ser Asn Asp Val Cys 1 5 10 15 Asn Arg ArgLys Cys His Lys Ala Leu Arg Gln Phe Phe Asp Lys Val 20 25 30 Pro Ala LysHis Ser Tyr Gly Met Leu Phe Cys Ser Cys Arg Asp Ile 35 40 45 Ala Cys ThrGlu Arg Arg Arg Gln Thr Ile Val Pro Val Cys Ser Tyr 50 55 60 Glu Glu ArgGlu Lys Pro Asn Cys Leu Asn Leu Gln Asp Ser Cys Lys 65 70 75 80 Thr AsnTyr Ile Cys Arg Ser Arg Leu Ala Asp Phe Phe Thr Asn Cys 85 90 95 Gln ProGlu Ser Arg Ser Val Ser Ser Cys Leu Lys Glu Asn Tyr Ala 100 105 110 AspCys Leu Leu Ala Tyr Ser Gly Leu Ile Gly Thr Val Met Thr Pro 115 120 125Asn Tyr Ile Asp Ser Ser Ser Leu Ser Val Ala Pro Trp Cys Asp Cys 130 135140 Ser Asn Ser Gly Asn Asp Leu Glu Glu Cys Leu Lys Phe Leu Asn Phe 145150 155 160 Phe Lys Asp Asn Thr Cys Leu Lys Asn Ala Ile Gln Ala Phe GlyAsn 165 170 175 Gly Ser Asp Val Thr Val Trp Gln Pro Ala Phe Pro Val GlnThr Thr 180 185 190 Thr Ala Ala Thr Thr Thr Ala Leu Arg Val Lys Asn LysPro Leu Gly 195 200 205 Pro Ala Gly Ser Glu Asn Glu Ile Pro Thr His ValLeu Pro Pro Cys 210 215 220 Ala Asn Leu Gln Ala Gln Lys Leu Lys Ser AsnVal Ser Gly Asn Thr 225 230 235 240 His Leu Cys Ile Ser Asn Gly Asn TyrGlu Lys Glu Gly Leu Gly Ala 245 250 255 Ser Ser His Ile Thr Thr Lys SerMet Ala Ala Pro Pro Ser Cys Gly 260 265 270 Leu Ser Pro Leu Leu Val LeuVal Val Thr Ala Leu Ser Thr Leu Leu 275 280 285 Ser Leu Thr Glu Thr Ser290 17 659 DNA HUMAN CDS (2)..(658) 17 g aat ttg cag gac tcc tgc aag acgaat tac atc tgc aga tct cgc ctt 49 Asn Leu Gln Asp Ser Cys Lys Thr AsnTyr Ile Cys Arg Ser Arg Leu 1 5 10 15 gcg gat ttt ttt acc aac tgc cagcca gag tca agg tct gtc agc agc 97 Ala Asp Phe Phe Thr Asn Cys Gln ProGlu Ser Arg Ser Val Ser Ser 20 25 30 tgt cta aag gaa aac tac gct gac tgcctc ctc gcc tac tcg ggg ctt 145 Cys Leu Lys Glu Asn Tyr Ala Asp Cys LeuLeu Ala Tyr Ser Gly Leu 35 40 45 att ggc aca gtc atg acc ccc aac tac atagac tcc agt agc ctc agt 193 Ile Gly Thr Val Met Thr Pro Asn Tyr Ile AspSer Ser Ser Leu Ser 50 55 60 gtg gcc cca tgg tgt gac tgc agc aac agt gggaac gac cta gaa gag 241 Val Ala Pro Trp Cys Asp Cys Ser Asn Ser Gly AsnAsp Leu Glu Glu 65 70 75 80 tgc ttg aaa ttt ttg aat ttc ttc aag gac aataca tgt ctt aaa aat 289 Cys Leu Lys Phe Leu Asn Phe Phe Lys Asp Asn ThrCys Leu Lys Asn 85 90 95 gca att caa gcc ttt ggc aat ggc tcc gat gtg accgtg tgg cag cca 337 Ala Ile Gln Ala Phe Gly Asn Gly Ser Asp Val Thr ValTrp Gln Pro 100 105 110 gcc ttc cca gta cag acc acc act gcc act acc accact gcc ctc cgg 385 Ala Phe Pro Val Gln Thr Thr Thr Ala Thr Thr Thr ThrAla Leu Arg 115 120 125 gtt aag aac aag ccc ctg ggg cca gca ggg tct gagaat gaa att ccc 433 Val Lys Asn Lys Pro Leu Gly Pro Ala Gly Ser Glu AsnGlu Ile Pro 130 135 140 act cat gtt ttg cca ccg tgt gca aat tta cag gcacag aag ctg aaa 481 Thr His Val Leu Pro Pro Cys Ala Asn Leu Gln Ala GlnLys Leu Lys 145 150 155 160 tcc aat gtg tcg ggc aat aca cac ctc tgt atttcc aat ggt aat tat 529 Ser Asn Val Ser Gly Asn Thr His Leu Cys Ile SerAsn Gly Asn Tyr 165 170 175 gaa aaa gaa ggt ctc ggt gct tcc agc cac ataacc aca aaa tca atg 577 Glu Lys Glu Gly Leu Gly Ala Ser Ser His Ile ThrThr Lys Ser Met 180 185 190 gct gct cct cca agc tgt ggt ctg agc cca ctgctg gtc ctg gtg gta 625 Ala Ala Pro Pro Ser Cys Gly Leu Ser Pro Leu LeuVal Leu Val Val 195 200 205 acc gct ctg tcc acc cta tta tct tta aca gaaa 659 Thr Ala Leu Ser Thr Leu Leu Ser Leu Thr Glu 210 215 18 219 PRTHUMAN misc_feature (1)..(659) Note= “1 to 659 is 1033 to 1691 of Figure5 Hsgr-21ar” 18 Asn Leu Gln Asp Ser Cys Lys Thr Asn Tyr Ile Cys Arg SerArg Leu 1 5 10 15 Ala Asp Phe Phe Thr Asn Cys Gln Pro Glu Ser Arg SerVal Ser Ser 20 25 30 Cys Leu Lys Glu Asn Tyr Ala Asp Cys Leu Leu Ala TyrSer Gly Leu 35 40 45 Ile Gly Thr Val Met Thr Pro Asn Tyr Ile Asp Ser SerSer Leu Ser 50 55 60 Val Ala Pro Trp Cys Asp Cys Ser Asn Ser Gly Asn AspLeu Glu Glu 65 70 75 80 Cys Leu Lys Phe Leu Asn Phe Phe Lys Asp Asn ThrCys Leu Lys Asn 85 90 95 Ala Ile Gln Ala Phe Gly Asn Gly Ser Asp Val ThrVal Trp Gln Pro 100 105 110 Ala Phe Pro Val Gln Thr Thr Thr Ala Thr ThrThr Thr Ala Leu Arg 115 120 125 Val Lys Asn Lys Pro Leu Gly Pro Ala GlySer Glu Asn Glu Ile Pro 130 135 140 Thr His Val Leu Pro Pro Cys Ala AsnLeu Gln Ala Gln Lys Leu Lys 145 150 155 160 Ser Asn Val Ser Gly Asn ThrHis Leu Cys Ile Ser Asn Gly Asn Tyr 165 170 175 Glu Lys Glu Gly Leu GlyAla Ser Ser His Ile Thr Thr Lys Ser Met 180 185 190 Ala Ala Pro Pro SerCys Gly Leu Ser Pro Leu Leu Val Leu Val Val 195 200 205 Thr Ala Leu SerThr Leu Leu Ser Leu Thr Glu 210 215 19 630 DNA HUMAN CDS (3)..(629) 19ac atc tgc aga tct cgc ctt gcg gat ttt ttt acc aac tgc cag cca 47 IleCys Arg Ser Arg Leu Ala Asp Phe Phe Thr Asn Cys Gln Pro 1 5 10 15 gagtca agg tct gtc agc agc tgt cta aag gaa aac tac gct gac tgc 95 Glu SerArg Ser Val Ser Ser Cys Leu Lys Glu Asn Tyr Ala Asp Cys 20 25 30 ctc ctcgcc tac tcg ggg ctt att ggc aca gtc atg acc ccc aac tac 143 Leu Leu AlaTyr Ser Gly Leu Ile Gly Thr Val Met Thr Pro Asn Tyr 35 40 45 ata gac tccagt agc ctc agt gtg gcc cca tgg tgt gac tgc agc aac 191 Ile Asp Ser SerSer Leu Ser Val Ala Pro Trp Cys Asp Cys Ser Asn 50 55 60 agt ggg aac gaccta gaa gag tgc ttg aaa ttt ttg aat ttc ttc aag 239 Ser Gly Asn Asp LeuGlu Glu Cys Leu Lys Phe Leu Asn Phe Phe Lys 65 70 75 gac aat aca tgt cttaaa aat gca att caa gcc ttt ggc aat ggc tcc 287 Asp Asn Thr Cys Leu LysAsn Ala Ile Gln Ala Phe Gly Asn Gly Ser 80 85 90 95 gat gtg acc gtg tggcag cca gcc ttc cca gta cag acc acc act gcc 335 Asp Val Thr Val Trp GlnPro Ala Phe Pro Val Gln Thr Thr Thr Ala 100 105 110 act acc acc act gccctc cgg gtt aag aac aag ccc ctg ggg cca gca 383 Thr Thr Thr Thr Ala LeuArg Val Lys Asn Lys Pro Leu Gly Pro Ala 115 120 125 ggg tct gag aat gaaatt ccc act cat gtt ttg cca ccg tgt gca aat 431 Gly Ser Glu Asn Glu IlePro Thr His Val Leu Pro Pro Cys Ala Asn 130 135 140 tta cag gca cag aagctg aaa tcc aat gtg tcg ggc aat aca cac ctc 479 Leu Gln Ala Gln Lys LeuLys Ser Asn Val Ser Gly Asn Thr His Leu 145 150 155 tgt att tcc aat ggtaat tat gaa aaa gaa ggt ctc ggt gct tcc agc 527 Cys Ile Ser Asn Gly AsnTyr Glu Lys Glu Gly Leu Gly Ala Ser Ser 160 165 170 175 cac ata acc acaaaa tca atg gct gct cct cca agc tgt ggt ctg agc 575 His Ile Thr Thr LysSer Met Ala Ala Pro Pro Ser Cys Gly Leu Ser 180 185 190 cca ctg ctg gtcctg gtg gta acc gct ctg tcc acc cta tta tct tta 623 Pro Leu Leu Val LeuVal Val Thr Ala Leu Ser Thr Leu Leu Ser Leu 195 200 205 aca gaa a 630Thr Glu 20 209 PRT HUMAN misc_feature (1)..(630) Note= “1 to 630 is 1062to 1691 of Figure 5 Hsgr-21br” 20 Ile Cys Arg Ser Arg Leu Ala Asp PhePhe Thr Asn Cys Gln Pro Glu 1 5 10 15 Ser Arg Ser Val Ser Ser Cys LeuLys Glu Asn Tyr Ala Asp Cys Leu 20 25 30 Leu Ala Tyr Ser Gly Leu Ile GlyThr Val Met Thr Pro Asn Tyr Ile 35 40 45 Asp Ser Ser Ser Leu Ser Val AlaPro Trp Cys Asp Cys Ser Asn Ser 50 55 60 Gly Asn Asp Leu Glu Glu Cys LeuLys Phe Leu Asn Phe Phe Lys Asp 65 70 75 80 Asn Thr Cys Leu Lys Asn AlaIle Gln Ala Phe Gly Asn Gly Ser Asp 85 90 95 Val Thr Val Trp Gln Pro AlaPhe Pro Val Gln Thr Thr Thr Ala Thr 100 105 110 Thr Thr Thr Ala Leu ArgVal Lys Asn Lys Pro Leu Gly Pro Ala Gly 115 120 125 Ser Glu Asn Glu IlePro Thr His Val Leu Pro Pro Cys Ala Asn Leu 130 135 140 Gln Ala Gln LysLeu Lys Ser Asn Val Ser Gly Asn Thr His Leu Cys 145 150 155 160 Ile SerAsn Gly Asn Tyr Glu Lys Glu Gly Leu Gly Ala Ser Ser His 165 170 175 IleThr Thr Lys Ser Met Ala Ala Pro Pro Ser Cys Gly Leu Ser Pro 180 185 190Leu Leu Val Leu Val Val Thr Ala Leu Ser Thr Leu Leu Ser Leu Thr 195 200205 Glu 21 1075 DNA HUMAN CDS (2)..(445) 21 t ggg aac gac cta gaa gagtgc ttg aaa ttt ttg aat ttc ttc aag gac 49 Gly Asn Asp Leu Glu Glu CysLeu Lys Phe Leu Asn Phe Phe Lys Asp 1 5 10 15 aat aca tgt ctt aaa aatgca att caa gcc ttt ggc aat ggc tcc gat 97 Asn Thr Cys Leu Lys Asn AlaIle Gln Ala Phe Gly Asn Gly Ser Asp 20 25 30 gtg acc gtg tgg cag cca gccttc cca gta cag acc acc act gcc act 145 Val Thr Val Trp Gln Pro Ala PhePro Val Gln Thr Thr Thr Ala Thr 35 40 45 acc acc act gcc ctc cgg gtt aagaac aag ccc ctg ggg cca gca ggg 193 Thr Thr Thr Ala Leu Arg Val Lys AsnLys Pro Leu Gly Pro Ala Gly 50 55 60 tct gag aat gaa att ccc act cat gttttg cca ccg tgt gca aat tta 241 Ser Glu Asn Glu Ile Pro Thr His Val LeuPro Pro Cys Ala Asn Leu 65 70 75 80 cag gca cag aag ctg aaa tcc aat gtgtcg ggc aat aca cac ctc tgt 289 Gln Ala Gln Lys Leu Lys Ser Asn Val SerGly Asn Thr His Leu Cys 85 90 95 att tcc aat ggt aat tat gaa aaa gaa ggtctc ggt gct tcc agc cac 337 Ile Ser Asn Gly Asn Tyr Glu Lys Glu Gly LeuGly Ala Ser Ser His 100 105 110 ata acc aca aaa tca atg gct gct cct ccaagc tgt ggt ctg agc cca 385 Ile Thr Thr Lys Ser Met Ala Ala Pro Pro SerCys Gly Leu Ser Pro 115 120 125 ctg ctg gtc ctg gtg gta acc gct ctg tccacc cta tta tct tta aca 433 Leu Leu Val Leu Val Val Thr Ala Leu Ser ThrLeu Leu Ser Leu Thr 130 135 140 gaa aca tca tag ctgcattaaa aaaatacaatatggacatgt aaaaagacaa 485 Glu Thr Ser 145 aaaccaagtt atctgtttcctgttctcttg tatagctgaa attccagttt aggagctcag 545 ttgagaaaca gttccattcaactggaacat tttttttttt ccttttaaga aagcttcttg 605 tgatccttcg gggcttctgtgaaaaacctg atgcagtgct ccatccaaac tcagaaggct 665 ttgggatatg ctgtattttaaagggacagt ttgtaacttg ggctgtaaag caaactgggg 725 ctgtgttttc gatgatgatgatcatcatga tcatgatnnn nnnnnnnnnn nnnnnnnnnn 785 nnnnnnnnnn nnnnnngattttaacagttt tacttctggc ctttcctagc tagagaagga 845 gttaatattt ctaaggtaactcccatatct cctttaatga cattgatttc taatgatata 905 aatttcagcc tacattgatgccaagctttt ttgccacaaa gaagattctt accaagagtg 965 ggctttgtgg aaacagctggtactgatgtt cacctttata tatgtactag cattttccac 1025 gctgatgttt atgtactgtaaacagttctg cactcttgta caaaagaaaa 1075 22 147 PRT HUMAN misc_feature(1)..(1075) Note= “1 to 1075 is 1255 to 2330 of Figure 5 Hsgr-2” 22 GlyAsn Asp Leu Glu Glu Cys Leu Lys Phe Leu Asn Phe Phe Lys Asp 1 5 10 15Asn Thr Cys Leu Lys Asn Ala Ile Gln Ala Phe Gly Asn Gly Ser Asp 20 25 30Val Thr Val Trp Gln Pro Ala Phe Pro Val Gln Thr Thr Thr Ala Thr 35 40 45Thr Thr Thr Ala Leu Arg Val Lys Asn Lys Pro Leu Gly Pro Ala Gly 50 55 60Ser Glu Asn Glu Ile Pro Thr His Val Leu Pro Pro Cys Ala Asn Leu 65 70 7580 Gln Ala Gln Lys Leu Lys Ser Asn Val Ser Gly Asn Thr His Leu Cys 85 9095 Ile Ser Asn Gly Asn Tyr Glu Lys Glu Gly Leu Gly Ala Ser Ser His 100105 110 Ile Thr Thr Lys Ser Met Ala Ala Pro Pro Ser Cys Gly Leu Ser Pro115 120 125 Leu Leu Val Leu Val Val Thr Ala Leu Ser Thr Leu Leu Ser LeuThr 130 135 140 Glu Thr Ser 145 23 1059 DNA HUMAN CDS (3)..(428) 23 agtgc ttg aaa ttt ttg aat ttc ttc aag gac aat aca tgt ctt aaa 47 Cys LeuLys Phe Leu Asn Phe Phe Lys Asp Asn Thr Cys Leu Lys 1 5 10 15 aat gcaatt caa gcc ttt ggc aat ggc tcc gat gtg acc gtg tgg cag 95 Asn Ala IleGln Ala Phe Gly Asn Gly Ser Asp Val Thr Val Trp Gln 20 25 30 cca gcc ttccca gta cag acc acc act gcc act acc acc act gcc ctc 143 Pro Ala Phe ProVal Gln Thr Thr Thr Ala Thr Thr Thr Thr Ala Leu 35 40 45 cgg gtt aag aacaag ccc ctg ggg cca gca ggg tct gag aat gaa att 191 Arg Val Lys Asn LysPro Leu Gly Pro Ala Gly Ser Glu Asn Glu Ile 50 55 60 ccc act cat gtt ttgcca ccg tgt gca aat tta cag gca cag aag ctg 239 Pro Thr His Val Leu ProPro Cys Ala Asn Leu Gln Ala Gln Lys Leu 65 70 75 aaa tcc aat gtg tcg ggcaat aca cac ctc tgt att tcc aat ggt aat 287 Lys Ser Asn Val Ser Gly AsnThr His Leu Cys Ile Ser Asn Gly Asn 80 85 90 95 tat gaa aaa gaa ggt ctcggt gct tcc agc cac ata acc aca aaa tca 335 Tyr Glu Lys Glu Gly Leu GlyAla Ser Ser His Ile Thr Thr Lys Ser 100 105 110 atg gct gct cct cca agctgt ggt ctg agc cca ctg ctg gtc ctg gtg 383 Met Ala Ala Pro Pro Ser CysGly Leu Ser Pro Leu Leu Val Leu Val 115 120 125 gta acc gct ctg tcc acccta tta tct tta aca gaa aca tca tag 428 Val Thr Ala Leu Ser Thr Leu LeuSer Leu Thr Glu Thr Ser 130 135 140 ctgcattaaa aaaatacaat atggacatgtaaaaagacaa aaaccaagtt atctgtttcc 488 tgttctcttg tatagctgaa attccagtttaggagctcag ttgagaaaca gttccattca 548 actggaacat tttttttttt tccttttaagaaagcttctt gtgatccttt ggggcttctg 608 tgaaaaacct gatgcagtgc tccatccaaactcagaaggc tttgggatat gctgtatttt 668 aaagggacag tttgtaactt gggctgtaaagcaaactggg gctgtgtttt cgatgatgat 728 gatgatcatg atgatgatca tcatgatcatgatgatgatc atcatgatca tgatgatgat 788 tttaacagtt ttacttctgg cctttcctagctagagaagg agttaatatt tctaaggtaa 848 ctcccatatc tcctttaatg acattgatttctaatgatat aaatttcagc ctacattgat 908 gccaagcttt tttgccacaa agaagattcttaccaagagt gggctttgtg gaaacagctg 968 gtactgatgt tcacctttat atatgtactagcattttcca cgctgatgtt tatgtactgt 1028 aaacagttct gcactcttgt acaaaagaaa a1059 24 141 PRT HUMAN misc_feature (1)..(1059) Note= “1 to 1059 is 1272to 2330 of Figure 5 Hsgr-9” 24 Cys Leu Lys Phe Leu Asn Phe Phe Lys AspAsn Thr Cys Leu Lys Asn 1 5 10 15 Ala Ile Gln Ala Phe Gly Asn Gly SerAsp Val Thr Val Trp Gln Pro 20 25 30 Ala Phe Pro Val Gln Thr Thr Thr AlaThr Thr Thr Thr Ala Leu Arg 35 40 45 Val Lys Asn Lys Pro Leu Gly Pro AlaGly Ser Glu Asn Glu Ile Pro 50 55 60 Thr His Val Leu Pro Pro Cys Ala AsnLeu Gln Ala Gln Lys Leu Lys 65 70 75 80 Ser Asn Val Ser Gly Asn Thr HisLeu Cys Ile Ser Asn Gly Asn Tyr 85 90 95 Glu Lys Glu Gly Leu Gly Ala SerSer His Ile Thr Thr Lys Ser Met 100 105 110 Ala Ala Pro Pro Ser Cys GlyLeu Ser Pro Leu Leu Val Leu Val Val 115 120 125 Thr Ala Leu Ser Thr LeuLeu Ser Leu Thr Glu Thr Ser 130 135 140 25 10 PRT HUMAN 25 Gln Ser CysSer Thr Lys Tyr Arg Thr Leu 1 5 10 26 10 PRT HUMAN 26 Cys Lys Arg GlyMet Lys Lys Glu Lys Asn 1 5 10 27 10 PRT HUMAN 27 Leu Leu Glu Asp SerPro Tyr Glu Pro Val 1 5 10 28 10 PRT RAT 28 Cys Ser Tyr Glu Glu Arg GluArg Pro Asn 1 5 10 29 14 PRT RAT 29 Pro Ala Pro Pro Val Gln Thr Thr ThrAla Thr Thr Thr Thr 1 5 10 30 21 DNA HUMAN 30 ctgtttgaat ttgcaggact c 2131 36 DNA HUMAN 31 ctcctctcta agcttctaac cacagcttgg aggagc 36 32 37 DNAHUMAN 32 ctcctctcta agcttctatg ggctcagacc acagctt 37 33 60 DNA HUMAN 33ctcctctcta agcttctact tgtcatcgtc gtccttgtag tcaccacagc ttggaggagc 60 3460 DNA HUMAN 34 ctcctctcta agcttctact tgtcatcgtc gtccttgtag tctggctcagaccacagctt 60 35 12 PRT Unknown Synthetic peptide sequence of arrestin35 Val Phe Glu Glu Phe Ala Arg Gln Asn Leu Lys Cys 1 5 10 36 8 PRTUnknown FLAG peptide sequence 36 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 373209 DNA HUMAN misc_feature (1091)..(1091) N in position 1091 indicatesany nucleic acid. 37 aatctggcct cggaacacgc cattctccgc gccgcttccaataaccacta acatccctaa 60 cgagcatccg agccgagggc tctgctcgga aatcgtcctggcccaactcg gcccttcgag 120 ctctcgaaga ttaccgcatc tatttttttt ttcttttttttcttttccta gcgcagataa 180 agtgagcccg gaaagggaag gagggggcgg ggacaccattgccctgaaag aataaataag 240 taaataaaca aactggctcc tcgccgcagc tggacgcggtcggttgagtc caggttgggt 300 cggacctgaa cccctaaaag cggaaccgcc tcccgccctcgccatcccgg agctgagtcg 360 ccggcggcgg tggctgctgc cagacccgga gtttcctctttcactggatg gagctgaact 420 ttgggcggcc agagcagcac agctgtccgg ggatcgctgcacgctgagct ccctcggcaa 480 gacccagcgg cggctcggga tttttttggg ggggcggggaccagccccgc gccggcacca 540 tgttcctggc gaccctgtac ttcgcgctgc cgctcttggacttgctcctg tcggccgaag 600 tgagcggcgg agaccgcctg gattgcgtga aagccagtgatcagtgcctg aaggagcaga 660 gctgcagcac caagtaccgc acgctaaggc agtgcgtggcgggcaaggag accaacttca 720 gcctggcatc cggcctggag gccaaggatg agtgccgcagcgccatggag gccctgaagc 780 agaagtcgct ctacaactgc cgctgcaagc ggggtatgaagaaggagaag aactgcctgc 840 gcatttactg gagcatgtac cagagcctgc agggaaatgatctgctggag gattccccat 900 atgaaccagt taacagcaga ttgtcagata tattccgggtggtcccattc atatcagatg 960 tttttcagca agtggagcac attcccaaag ggaacaactgcctggatgca gcgaaggcct 1020 gcaacctcga cgacatttgc aagaagtaca ggtcggcgtacatcaccccg tgcaccacca 1080 gcgtgtccaa ngatgtctgc aaccgccgca agtgccacaaggccctccgg cagttctttg 1140 acaaggtccc ggccaagcac agctacggaa tgctcttctgctcctgccgg gacatcgcct 1200 gcacagagcg gaggcgacag accatcgtgc ctgtgtgctcctatgaagag agggagaagc 1260 ccaactgttt gaatttgcag gactcctgca agacgaattacatctgcaga tctcgccttg 1320 cggatttttt taccaactgc cagccagagt caaggtctgtcagcagctgt ctaaaggaaa 1380 actacgctga ctgcctcctc gcctactcgg ggcttattggcacagtcatg acccccaact 1440 acatagactc cagtagcctc agtgtggccc catggtgtgactgcagcaac agtgggaacg 1500 acctagaaga gtgcttgaaa tttttgaatt tcttcaaggacaatacatgt cttaaaaatg 1560 caattcaagc ctttggcaat ggctccgatg tgaccgtgtggcagccagcc ttcccagtac 1620 agaccaccac tgccactacc accactgccc tccgggttaagaacaagccc ctggggccag 1680 cagggtctga gaatgaaatt cccactcatg ttttgccaccgtgtgcaaat ttacaggcac 1740 agaagctgaa atccaatgtg tcgggcaata cacacctctgtatttccaat ggtaattatg 1800 aaaaagaagg tctcggtgct tccagccaca taaccacaaaatcaatggct gctcctccaa 1860 gctgtggtct gagcccactg ctggtcctgg tggtaaccgctctgtccacc ctattatctt 1920 taacagaaac atcatagctg cattaaaaaa atacaatatggacatgtaaa aagacaaaaa 1980 ccaagttatc tgtttcctgt tctcttgtat agctgaaattccagtttagg agctcagttg 2040 agaaacagtt ccattcaact ggaacatttt tttttttnccttttaagaaa gcttcttgtg 2100 atccttcggg gcttctgtga aaaacctgat gcagtgctccatccaaactc agaaggcttt 2160 gggatatgct gtattttaaa gggacagttt gtaacttgggctgtaaagca aactggggct 2220 gtgttttcga tgatgatgat catcatgatc atgatnnnnnnnnnnnnnnn nnnnnnnnnn 2280 nnnnnnnnnn nnnngatttt aacagtttta cttctggcctttcctagcta gagaaggagt 2340 taatatttct aaggtaactc ccatatctcc tttaatgacattgatttcta atgatataaa 2400 tttcagccta cattgatgcc aagctttttt gccacaaagaagattcttac caagagtggg 2460 ctttgtggaa acagctggta ctgatgttca cctttatatatgtactagca ttttccacgc 2520 tgatgtttat gtactgtaaa cagttctgca ctcttgtacaaaagaaaaaa cacctgtcac 2580 atccaaatat agtatctgtc ttttcgtcaa aatagagagtggggaatgag tgtgccgatt 2640 caatacctca atccctgaac gacactctcc taatcctaagccttacctga gtgagaagcc 2700 ctttacctaa caaaagtcca atatagctga aatgtcgctctaatactctt tacacatatg 2760 aggttatatg tagaaaaaaa ttttactact aaatgatttcaactattggc tttctatatt 2820 ttgaaagtaa tgatattgtc tcattttttt actgatggtttaatacaaaa tacacagagc 2880 ttgtttcccc tcataagtag tgttcgctct gatatgaacttcacaaatac agctcatcaa 2940 aagcagactc tgagaagcct cgtgctgtag cagaaagttctgcatcatgt gactgtggac 3000 aggcaggagg aaacagaaca gacaagcatt gtcttttgtcattgctcgaa gtgcaagcgt 3060 gcatacctgt ggagggaact ggtggctgct tgtaaatgttctgcagcatc tcttgacaca 3120 cttgtcatga cacaatccag taccttggtt ttcaggttatctgacaaagg cagctttgat 3180 tgggacatgg aggcatgggc aggccggaa 3209 38 508DNA HUMAN 38 tctggcctcg gaacacgcca ttctccgcgc cgcttccaat aaccactaacatccctaacg 60 agcatccgag ccgagggctc tgctcggaaa tcgtcctggc ccaactcggcccttcgagct 120 ctcgaagatt accgcatcta tttttttttt cttttttttc ttttcctagcgcagataaag 180 tgagcccgga aagggaagga gggggcgggg acaccattgc cctgaaagaataaataagta 240 aataaacaaa ctggctcctc gccgcagctg gacgcggtcg gttgagtccaggttgggtcg 300 gacctgaacc cctaaaagcg gaaccgcctc ccgccctcgc catcccggagctgagtcgcc 360 ggcggcggtg gctgctgcca gacccggagt ttcctctttc actggatggagctgaacttt 420 gggcggccag agcagcacag ctgtccgggg atcgctgcac gctgagctccctcggcaaga 480 cccagcggcg gctcgggatt tttttggg 508 39 510 DNA HUMAN 39aatctggcct cggaacacgc cattctccgc gccgcttcca ataaccacta acatccctaa 60cgagcatccg agccgagggc tctgctcgga aatcgtcctg gcccaactcg gcccttcgag 120ctctcgaaga ttaccgcatc tatttttttt ttcttttttt tcttttccta gcgcagataa 180agtgagcccg gaaagggaag gagggggcgg ggacaccatt gccctgaaag aataaataag 240taaataaaca aactggctcc tcgccgcagc tggacgcggt cggttgagtc caggttgggt 300cggacctgaa cccctaaaag cggaaccgcc tcccgccctc gccatcccgg agctgagtcg 360ccggcggcgg tggctgctgc cagacccgga gtttcctctt tcactggatg gagctgaact 420ttgggcggcc agagcagcac agctgtccgg ggatcgctgc acgctgagct ccctcggcaa 480gacccagcgg cggctcggga tttttttggg 510 40 1927 DNA HUMAN misc_feature(550)..(550) N in position 550 indicates any nucleic acid 40 tctggcctcggaacacgcca ttctccgcgc cgcttccaat aaccactaac atccctaacg 60 agcatccgagccgagggctc tgctcggaaa tcgtcctggc ccaactcggc ccttcgagct 120 ctcgaagattaccgcatcta tttttttttt cttttttttc ttttcctagc gcagataaag 180 tgagcccggaaagggaagga gggggcgggg acaccattgc cctgaaagaa taaataagta 240 aataaacaaactggctcctc gccgcagctg gacgcggtcg gttgagtcca ggttgggtcg 300 gacctgaacccctaaaagcg gaaccgcctc ccgccctcgc catcccggag ctgagtcgcc 360 ggcggcggtggctgctgcca gacccggagt ttcctctttc actggatgga gctgaacttt 420 gggcggccagagcagcacag ctgtccgggg atcgctgcac gctgagctcc ctcggcaaga 480 cccagcggcggctcgggatt tttttggggg ggcggggacc agccccgcgc cggcaccatg 540 ttcctggcgnccctgtactt cgcgctgccg ctcttggact tgctcctgtc ggccgaagtg 600 agcggcggagaccgcctgga ttgcgtgaaa gccagtgatc agtgcctgaa ggagcagagc 660 tgcagcaccaagtaccgcac gctaaggcag tgcgtggcgg gcaaggagac caacttcagc 720 ctggcatccggcctggaggc caaggatgag tgccgcagcg ccatggaggc cctgaagcag 780 aagtcgctctacaactgccg ctgcaagcgg ggtatgaaga aggagaagaa ctgcctgcgc 840 atttactggagcatgtacca gagcctgcag ggaaatgatc tgctggagga ttccccatat 900 gaaccagttaacagcagatt gtcagatata ttccgggtgg tcccattcat atcagatgtt 960 tttcagcaagtggagcacat tcccaaaggg aacaactgcc tggatgcagc gaaggcctgc 1020 aacctcgacgacatttgcaa gaagtacagg tcggcgtaca tcaccccgtg caccaccagc 1080 gtgtccaacgatgtctgcaa ccgccgcaag tgccacaagg ccctccggca gttctttgac 1140 aaggtcccggccaagcacag ctacggaatg ctcttctgct cctgccggga catcgcctgc 1200 acagagcggaggcgacagac catcgtgcct gtgtgctcct atgaagagag ggagaagccc 1260 aactgtttgaatttgcagga ctcctgcaag acgaattaca tctgcagatc tcgccttgcg 1320 gatttttttaccaactgcca gccagagtca aggtctgtca gcagctgtct aaaggaaaac 1380 tacgctgactgcctcctcgc ctactcgggg cttattggca cagtcatgac ccccaactac 1440 atagactccagtagcctcag tgtggcccca tggtgtgact gcagcaacag tgggaacgac 1500 ctagaagagtgcttgaaatt tttgaatttc ttcaaggaca atacatgtct taaaaatgca 1560 attcaagcctttggcaatgg ctccgatgtg accgtgtggc agccagcctt cccagtacag 1620 accaccactgccactaccac cactgccctc cgggttaaga acaagcccct ggggccagca 1680 gggtctgagaatgaaattcc cactcatgtt ttgccaccgt gtgcaaattt acaggcacag 1740 aagctgaaatccaatgtgtc gggcaataca cacctctgta tttccaatgg taattatgaa 1800 aaagaaggtctcggtgcttc cagccacata accacaaaat caatggctgc tcctccaagc 1860 tgtggtctgagcccactgct ggtcctggtg gtaaccgctc tgtccaccct attatcttta 1920 acagaaa 192741 1929 DNA HUMAN 41 aatctggcct cggaacacgc cattctccgc gccgcttccaataaccacta acatccctaa 60 cgagcatccg agccgagggc tctgctcgga aatcgtcctggcccaactcg gcccttcgag 120 ctctcgaaga ttaccgcatc tatttttttt ttcttttttttcttttccta gcgcagataa 180 agtgagcccg gaaagggaag gagggggcgg ggacaccattgccctgaaag aataaataag 240 taaataaaca aactggctcc tcgccgcagc tggacgcggtcggttgagtc caggttgggt 300 cggacctgaa cccctaaaag cggaaccgcc tcccgccctcgccatcccgg agctgagtcg 360 ccggcggcgg tggctgctgc cagacccgga gtttcctctttcactggatg gagctgaact 420 ttgggcggcc agagcagcac agctgtccgg ggatcgctgcacgctgagct ccctcggcaa 480 gacccagcgg cggctcggga tttttttggg ggggcggggaccagccccgc gccggcacca 540 tgttcctggc gaccctgtac ttcgcgctgc cgctcttggacttgctcctg tcggccgaag 600 tgagcggcgg agaccgcctg gattgcgtga aagccagtgatcagtgcctg aaggagcaga 660 gctgcagcac caagtaccgc acgctaaggc agtgcgtggcgggcaaggag accaacttca 720 gcctggcatc cggcctggag gccaaggatg agtgccgcagcgccatggag gccctgaagc 780 agaagtcgct ctacaactgc cgctgcaagc ggggtatgaagaaggagaag aactgcctgc 840 gcatttactg gagcatgtac cagagcctgc agggaaatgatctgctggag gattccccat 900 atgaaccagt taacagcaga ttgtcagata tattccgggtggtcccattc atatcagatg 960 tttttcagca agtggagcac attcccaaag ggaacaactgcctggatgca gcgaaggcct 1020 gcaacctcga cgacatttgc aagaagtaca ggtcggcgtacatcaccccg tgcaccacca 1080 gcgtgtccaa cgatgtctgc aaccgccgca agtgccacaaggccctccgg cagttctttg 1140 acaaggtccc ggccaagcac agctacggaa tgctcttctgctcctgccgg gacatcgcct 1200 gcacagagcg gaggcgacag accatcgtgc ctgtgtgctcctatgaagag agggagaagc 1260 ccaactgttt gaatttgcag gactcctgca agacgaattacatctgcaga tctcgccttg 1320 cggatttttt taccaactgc cagccagagt caaggtctgtcagcagctgt ctaaaggaaa 1380 actacgctga ctgcctcctc gcctactcgg ggcttattggcacagtcatg acccccaact 1440 acatagactc cagtagcctc agtgtggccc catggtgtgactgcagcaac agtgggaacg 1500 acctagaaga gtgcttgaaa tttttgaatt tcttcaaggacaatacatgt cttaaaaatg 1560 caattcaagc ctttggcaat ggctccgatg tgaccgtgtggcagccagcc ttcccagtac 1620 agaccaccac tgccactacc accactgccc tccgggttaagaacaagccc ctggggccag 1680 cagggtctga gaatgaaatt cccactcatg ttttgccaccgtgtgcaaat ttacaggcac 1740 agaagctgaa atccaatgtg tcgggcaata cacacctctgtatttccaat ggtaattatg 1800 aaaaagaagg tctcggtgct tccagccaca taaccacaaaatcaatggct gctcctccaa 1860 gctgtggtct gagcccactg ctggtcctgg tggtaaccgctctgtccacc ctattatctt 1920 taacagaaa 1929 42 699 DNA HUMAN 42 gtcggcgtacatcaccccgt gcaccaccag cgtgtccaat gatgtctgca accgccgcaa 60 gtgccacaaggccctccggc agttctttga caaggtcccg gccaagcaca gctacggaat 120 gctcttctgctcctgccggg acatcgcctg cacagagcgg aggcgacaga ccatcgtgcc 180 tgtgtgctcctatgaagaga gggagaagcc caactgtttg aatttgcagg actcctgcaa 240 gacgaattacatctgcagat ctcgccttgc ggattttttt accaactgcc agccagagtc 300 aaggtctgtcagcagctgtc taaaggaaaa ctacgctgac tgcctcctcg cctactcggg 360 gcttattggcacagtcatga cccccaacta catagactcc agtagcctca gtgtggcccc 420 atggtgtgactgcagcaaca gtgggaacga cctagaagag tgcttgaaat ttttgaattt 480 cttcaaggacaatacatgtc ttaaaaatgc aattcaagcc tttggcaatg gctccgatgt 540 gaccgtgtggcagccagcct tcccagtaca gaccaccact gccgctacca ccactgccct 600 ccgggttaagaacaagcccc tggggccagc agggtctgag aatgaaattc ccactcatgt 660 tttgccaccgtgtgcaaatt tacaggcaca gaagctgaa 699 43 2158 DNA HUMAN misc_feature(1027)..(1027) N in position 1027 indicates a position of divergencebetween different receptor clones. 43 gtcggcgtac atcaccccgt gcaccaccagcgtgtccaat gatgtctgca accgccgcaa 60 gtgccacaag gccctccggc agttctttgacaaggtcccg gccaagcaca gctacggaat 120 gctcttctgc tcctgccggg acatcgcctgcacagagcgg aggcgacaga ccatcgtgcc 180 tgtgtgctcc tatgaagaga gggagaagcccaactgtttg aatttgcagg actcctgcaa 240 gacgaattac atctgcagat ctcgccttgcggattttttt accaactgcc agccagagtc 300 aaggtctgtc agcagctgtc taaaggaaaactacgctgac tgcctcctcg cctactcggg 360 gcttattggc acagtcatga cccccaactacatagactcc agtagcctca gtgtggcccc 420 atggtgtgac tgcagcaaca gtgggaacgacctagaagag tgcttgaaat ttttgaattt 480 cttcaaggac aatacatgtc ttaaaaatgcaattcaagcc tttggcaatg gctccgatgt 540 gaccgtgtgg cagccagcct tcccagtacagaccaccact gccgctacca ccactgccct 600 ccgggttaag aacaagcccc tggggccagcagggtctgag aatgaaattc ccactcatgt 660 tttgccaccg tgtgcaaatt tacaggcacagaagctgaaa tccaatgtgt cgggcaatac 720 acacctctgt atttccaatg gtaattatgaaaaagaaggt ctcggtgctt ccagccacat 780 aaccacaaaa tcaatggctg ctcctccaagctgtggtctg agcccactgc tggtcctggt 840 ggtaaccgct ctgtccaccc tattatctttaacagaaaca tcatagctgc attaaaaaaa 900 tacaatatgg acatgtaaaa agacaaaaaccaagttatct gtttcctgtt ctcttgtata 960 gctgaaattc cagtttagga gctcagttgagaaacagttc cattcaactg gaacattttt 1020 ttttttncct tttaagaaag cttcttgtgatccttcgggg cttctgtgaa aaacctgatg 1080 cagtgctcca tccaaactca gaaggctttgggatatgctg tattttaaag ggacagtttg 1140 taacttgggc tgtaaagcaa actggggctgtgttttcgat gatgatgatc atcatgatca 1200 tgatnnnnnn nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnngatttta acagttttac 1260 ttctggcctt tcctagctag agaaggagttaatatttcta aggtaactcc catatctcct 1320 ttaatgacat tgatttctaa tgatataaatttcagcctac attgatgcca agcttttttg 1380 ccacaaagaa gattcttacc aagagtgggctttgtggaaa cagctggtac tgatgttcac 1440 ctttatatat gtactagcat tttccacgctgatgtttatg tactgtaaac agttctgcac 1500 tcttgtacaa aagaaaaaac acctgtcacatccaaatata gtatctgtct tttcgtcaaa 1560 atagagagtg gggaatgagt gtgccgattcaatacctcaa tccctgaacg acactctcct 1620 aatcctaagc cttacctgag tgagaagccctttacctaac aaaagtccaa tatagctgaa 1680 atgtcgctct aatactcttt acacatatgaggttatatgt agaaaaaaat tttactacta 1740 aatgatttca actattggct ttctatattttgaaagtaat gatattgtct cattttttta 1800 ctgatggttt aatacaaaat acacagagcttgtttcccct cataagtagt gttcgctctg 1860 atatgaactt cacaaataca gctcatcaaaagcagactct gagaagcctc gtgctgtagc 1920 agaaagttct gcatcatgtg actgtggacaggcaggagga aacagaacag acaagcattg 1980 tcttttgtca ttgctcgaag tgcaagcgtgcatacctgtg gagggaactg gtggctgctt 2040 gtaaatgttc tgcagcatct cttgacacacttgtcatgac acaatccagt accttggttt 2100 tcaggttatc tgacaaaggc agctttgattgggacatgga ggcatgggca ggccggaa 2158 44 659 DNA HUMAN 44 gaatttgcaggactcctgca agacgaatta catctgcaga tctcgccttg cggatttttt 60 taccaactgccagccagagt caaggtctgt cagcagctgt ctaaaggaaa actacgctga 120 ctgcctcctcgcctactcgg ggcttattgg cacagtcatg acccccaact acatagactc 180 cagtagcctcagtgtggccc catggtgtga ctgcagcaac agtgggaacg acctagaaga 240 gtgcttgaaatttttgaatt tcttcaagga caatacatgt cttaaaaatg caattcaagc 300 ctttggcaatggctccgatg tgaccgtgtg gcagccagcc ttcccagtac agaccaccac 360 tgccactaccaccactgccc tccgggttaa gaacaagccc ctggggccag cagggtctga 420 gaatgaaattcccactcatg ttttgccacc gtgtgcaaat ttacaggcac agaagctgaa 480 atccaatgtgtcgggcaata cacacctctg tatttccaat ggtaattatg aaaaagaagg 540 tctcggtgcttccagccaca taaccacaaa atcaatggct gctcctccaa gctgtggtct 600 gagcccactgctggtcctgg tggtaaccgc tctgtccacc ctattatctt taacagaaa 659 45 630 DNAHUMAN 45 acatctgcag atctcgcctt gcggattttt ttaccaactg ccagccagagtcaaggtctg 60 tcagcagctg tctaaaggaa aactacgctg actgcctcct cgcctactcggggcttattg 120 gcacagtcat gacccccaac tacatagact ccagtagcct cagtgtggccccatggtgtg 180 actgcagcaa cagtgggaac gacctagaag agtgcttgaa atttttgaatttcttcaagg 240 acaatacatg tcttaaaaat gcaattcaag cctttggcaa tggctccgatgtgaccgtgt 300 ggcagccagc cttcccagta cagaccacca ctgccactac caccactgccctccgggtta 360 agaacaagcc cctggggcca gcagggtctg agaatgaaat tcccactcatgttttgccac 420 cgtgtgcaaa tttacaggca cagaagctga aatccaatgt gtcgggcaatacacacctct 480 gtatttccaa tggtaattat gaaaaagaag gtctcggtgc ttccagccacataaccacaa 540 aatcaatggc tgctcctcca agctgtggtc tgagcccact gctggtcctggtggtaaccg 600 ctctgtccac cctattatct ttaacagaaa 630 46 1076 DNA HUMANmisc_feature (586)..(586) N in position 586 indicates a position ofdivergence between different receptor clones. 46 tgggaacgac ctagaagagtgcttgaaatt tttgaatttc ttcaaggaca atacatgtct 60 taaaaatgca attcaagcctttggcaatgg ctccgatgtg accgtgtggc agccagcctt 120 cccagtacag accaccactgccactaccac cactgccctc cgggttaaga acaagcccct 180 ggggccagca gggtctgagaatgaaattcc cactcatgtt ttgccaccgt gtgcaaattt 240 acaggcacag aagctgaaatccaatgtgtc gggcaataca cacctctgta tttccaatgg 300 taattatgaa aaagaaggtctcggtgcttc cagccacata accacaaaat caatggctgc 360 tcctccaagc tgtggtctgagcccactgct ggtcctggtg gtaaccgctc tgtccaccct 420 attatcttta acagaaacatcatagctgca ttaaaaaaat acaatatgga catgtaaaaa 480 gacaaaaacc aagttatctgtttcctgttc tcttgtatag ctgaaattcc agtttaggag 540 ctcagttgag aaacagttccattcaactgg aacatttttt tttttncctt ttaagaaagc 600 ttcttgtgat ccttcggggcttctgtgaaa aacctgatgc agtgctccat ccaaactcag 660 aaggctttgg gatatgctgtattttaaagg gacagtttgt aacttgggct gtaaagcaaa 720 ctggggctgt gttttcgatgatgatgatca tcatgatcat gatnnnnnnn nnnnnnnnnn 780 nnnnnnnnnn nnnnnnnnnnnngattttaa cagttttact tctggccttt cctagctaga 840 gaaggagtta atatttctaaggtaactccc atatctcctt taatgacatt gatttctaat 900 gatataaatt tcagcctacattgatgccaa gcttttttgc cacaaagaag attcttacca 960 agagtgggct ttgtggaaacagctggtact gatgttcacc tttatatatg tactagcatt 1020 ttccacgctg atgtttatgtactgtaaaca gttctgcact cttgtacaaa agaaaa 1076 47 1059 DNA HUMAN 47agtgcttgaa atttttgaat ttcttcaagg acaatacatg tcttaaaaat gcaattcaag 60cctttggcaa tggctccgat gtgaccgtgt ggcagccagc cttcccagta cagaccacca 120ctgccactac caccactgcc ctccgggtta agaacaagcc cctggggcca gcagggtctg 180agaatgaaat tcccactcat gttttgccac cgtgtgcaaa tttacaggca cagaagctga 240aatccaatgt gtcgggcaat acacacctct gtatttccaa tggtaattat gaaaaagaag 300gtctcggtgc ttccagccac ataaccacaa aatcaatggc tgctcctcca agctgtggtc 360tgagcccact gctggtcctg gtggtaaccg ctctgtccac cctattatct ttaacagaaa 420catcatagct gcattaaaaa aatacaatat ggacatgtaa aaagacaaaa accaagttat 480ctgtttcctg ttctcttgta tagctgaaat tccagtttag gagctcagtt gagaaacagt 540tccattcaac tggaacattt tttttttttc cttttaagaa agcttcttgt gatcctttgg 600ggcttctgtg aaaaacctga tgcagtgctc catccaaact cagaaggctt tgggatatgc 660tgtattttaa agggacagtt tgtaacttgg gctgtaaagc aaactggggc tgtgttttcg 720atgatgatga tgatcatgat gatgatcatc atgatcatga tgatgatcat catgatcatg 780atgatgattt taacagtttt acttctggcc tttcctagct agagaaggag ttaatatttc 840taaggtaact cccatatctc ctttaatgac attgatttct aatgatataa atttcagcct 900acattgatgc caagcttttt tgccacaaag aagattctta ccaagagtgg gctttgtgga 960aacagctggt actgatgttc acctttatat atgtactagc attttccacg ctgatgttta 1020tgtactgtaa acagttctgc actcttgtac aaaagaaaa 1059

What is claimed is:
 1. An isolated and purified protein comprising anamino acid sequence as depicted in FIGS. 2 or 4 (SEQ ID NO: 2 or 4) andanalogs thereof wherein the protein is capable of complexing with glialcell line-derived neurotrophic factor (GDNF) and thereby mediating cellresponse to GDNF.
 2. A protein of claim 1 comprising the amino acidsequence as depicted in FIG. 2 (SEQ ID NO: 2).
 3. A protein of claim 1comprising the amino acid sequence as depicted in FIG. 4 (SEQ ID NO:4).4. A protein of claim 1 comprising the amino acid sequence Ser¹⁸ throughPro⁴⁴⁶ as depicted in FIG. 2 (SEQ ID NO:2).
 5. A protein of claim 1comprising the amino acid sequence Asp²⁵ through Leu⁴⁴⁷ as depicted inFIG. 2 (SEQ ID NO:2).
 6. A protein of claim 1 comprising the amino acidsequence Cys²⁹ through Cys⁴⁴² as depicted in FIG. 2 (SEQ ID NO:2).
 7. Aprotein of claim 1 comprising the amino acid sequence Ala¹⁹ throughVal⁴⁵⁰ as depicted in FIG. 4 (SEQ ID NO:4).
 8. A protein of claim 1comprising the amino acid sequence Cys²⁹ through Cys⁴⁴³ as depicted inFIG. 4 (SEQ ID NO:4).
 9. A protein of claim 1 which is glycosylated. 10.A protein of claim 1 which is non-glycosylated.
 11. A protein of claims1 to 10 which is produced by recombinant technology or chemicalsynthesis.
 12. A pharmaceutical composition comprising a protein asclaimed in any one of claims 1 to 10 in combination with apharmaceutically acceptable carrier.
 13. An isolated nucleic acidsequence encoding a neurotrophic factor receptor protein comprising anamino acid sequence as claimed in any one of claims 1 to
 8. 14. Anisolated nucleic acid sequence encoding a neurotrophic factor receptorprotein comprising an amino acid sequence as depicted in FIGS. 2 or 4(SEQ ID NO: 2 or 4) and analogs thereof wherein the protein is capableof complexing with glial cell line-derived neurotrophic factor (GDNF)and thereby mediating cell response to GDNF.
 15. A nucleic acid sequenceof claim 14 encoding a neurotrophic factor receptor protein comprisingthe amino acid sequence as depicted in FIG. 2 (SEQ ID NO: 2).
 16. Anucleic acid sequence of claim 14 encoding a neurotrophic factorreceptor protein comprising the amino acid sequence as depicted in FIG.4 (SEQ ID NO:4).
 17. An isolated nucleic acid sequence comprising: (a) asequence set forth in FIG. 1 (SEQ ID NO: 1) comprising nucleotidesencoding Met¹ through Ser⁴⁶⁵ or FIG. 3 (SEQ ID NO: 3) comprisingnucleotides encoding Met¹ through Ser⁴⁶⁸, wherein said sequence encodesa neurotrophic factor receptor protein (GDNFR) capable of complexingwith glial cell line-derived neurotrophic factor (GDNF) and therebymediating cell response to GDNF; (b) a nucleic acid sequence which (1)hybridizes to a complementary sequence of (a) and (2) encodes an aminoacid sequence with GDNFR activity; and (c) a nucleic acid sequence whichbut for the degeneracy of the genetic code would hybridize to acomplementary sequence of (a) and (2) encodes an amino acid sequencewith GDNFR activity.
 18. A vector comprising a nucleic acid sequenceaccording to any of claims 14 to 17 operatively linked to one or moreoperational elements capable of effecting the amplification orexpression of said nucleic acid sequence.
 19. A vector comprising anucleic acid sequence encoding a neurotrophic factor receptor proteincomprising the amino acid sequence as depicted in FIGS. 2 or 4 (SEQ IDNO: 2 or 4) operatively linked to one or more operational elementscapable of effecting the amplification or expression of said nucleicacid sequence.
 20. A host cell transformed or transfected with thevector of claim
 18. 21. A host cell transformed or transfected with thevector of claim
 19. 22. A host cell of claim 20 selected from the groupconsisting of mammalian cells and bacterial cells.
 23. A host cell ofclaim 22 which is a COS-7 cell or E. coli.
 24. A host cell of claim 20wherein said cell is suitable for human implantation and wherein saidcell expresses and secretes said neurotrophic factor receptor.
 25. Ahost cell of claim 21 wherein said cell is suitable for humanimplantation and wherein said cell expresses and secretes saidneurotrophic factor receptor.
 26. A host cell of claim 20 wherein saidcell is transformed or transfected ex vivo.
 27. A host cell of claim 20wherein said cell is enclosed in a semipermeable membrane suitable forhuman implantation.
 28. A method for the production of a neurotrophicfactor receptor protein comprising the steps of: (a) culturing a hostcell, containing a nucleic acid sequence encoding a neurotrophic factorreceptor protein comprising an amino acid sequence as depicted in FIGS.2 or 4 (SEQ ID NO: 2 or 4) and analogs thereof wherein the protein iscapable of complexing with glial cell line-derived neurotrophic factor(GDNF) and thereby mediating cell response to GDNF, under conditionssuitable for the expression of said neurotrophic factor receptor proteinby said host cell; and (b) optionally, isolating said neurotrophicfactor receptor protein expressed by said host cell.
 29. A method ofclaim 28, wherein said nucleic acid sequence encodes a neurotrophicfactor receptor protein comprising the amino acid sequence as depictedin FIG. 2 (SEQ ID NO:2).
 30. A method of claim 28, wherein said nucleicacid sequence encodes a neurotrophic factor receptor protein comprisingthe amino acid sequence as depicted in FIG. 4 (SEQ ID NO:4).
 31. Amethod for the production of a neurotrophic factor receptor proteincomprising the steps of: (a) culturing a host cell transformed ortransfected with a nucleic acid sequence according to claim 17 underconditions suitable for the expression of said neurotrophic factorreceptor protein by said host cell; and (b) optionally, isolating saidneurotrophic factor receptor protein expressed by said host cell.
 32. Amethod of claim 28 or 31, further comprising the step of refolding theisolated neurotrophic factor receptor.
 33. A method of claim 28 or 31,wherein said host cell is a prokaryotic cell.
 34. A method of claim 28or 31, wherein said host cell is a eukaryotic cell.
 35. A substantiallypurified neurotrophic factor receptor protein prepared according to themethod of any of claims 28 to
 31. 36. The use of the neurotrophic factorreceptor protein of claim 1 for the manufacture of a pharmaceuticalcomposition.
 37. A method of treating improperly functioningdopaminergic nerve cells by administering a neurotrophic factor receptorprotein of claim
 1. 38. A method of treating Parkinson's disease byadministering a neurotrophic factor receptor protein of claim
 1. 39. Amethod of treating Alzheimer's disease by administering a neurotrophicfactor receptor protein of claim
 1. 40. A method of treating amyotrophiclateral sclerosis by administering a neurotrophic protein of claim 1.41. An antibody that binds to a neurotrophic factor receptor proteincomprising an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4.
 42. Theantibody of claim 41 wherein said antibody is a monoclonal antibody. 43.The antibody of claim 41 wherein said antibody is a polyclonal antibody.44. An antibody produced by immunizing an animal with a neurotrophicfactor receptor protein comprising an amino acid sequence of SEQ ID NO:2or SEQ ID NO:4.
 45. A hybridoma that produces a monoclonal antibody thatbinds to a neurotrophic factor receptor protein comprising an amino acidsequence of SEQ ID NO:2 or SEQ ID NO:4.
 46. A device for treating nervedamage, comprising: (a) a semipermeable membrane suitable forimplantation; and (b) cells encapsulated within said membrane, whereinsaid cells secrete a neurotrophic factor receptor protein according toclaim 1; said membrane being permeable to the neurotrophic factorreceptor protein and impermeable to materials detrimental to said cells.47. The device of claim 46, wherein said cells are naturally occurringcells that secrete said neurotrophic factor receptor protein.
 48. Thedevice of claim 46, wherein said cells have been modified to secretesaid neurotrophic factor receptor protein by means of a nucleic acidsequence comprising: (a) a sequence set forth in FIG. 1 (SEQ ID NO.: 1)comprising nucleotides encoding Met¹ through Ser⁴⁶⁵ or FIG. 3 (SEQ IDNO: 3) comprising nucleotides encoding Met¹ through Ser⁴⁶⁸ encoding aneurotrophic factor receptor protein (GDNFR) capable of complexing withglial cell line-derived neurotrophic factor (GDNF) and mediating cellresponse to GDNF; (b) a nucleic acid sequence which (1) hybridizes to acomplementary sequence of (a) and (2) encodes an amino acid sequencewith GDNFR activity; and (c) a nucleic acid sequence which but for thedegeneracy of the genetic code would hybridize to a complementarysequence of (a) and (2) encodes an amino acid sequence with GDNFRactivity.
 49. An assay device for analyzing a test sample for thepresence of glial cell line-derived neurotrophic factor, comprising: asolid phase containing or coated with a GDNFR protein, wherein saidGDNFR protein reacts with GDNF present in the test sample and produces adetectable reaction product indicative of the presence of GDNF.
 50. Amethod for analyzing a test sample for the presence of glial cellline-derived neurotrophic factor, comprising: contacting the sample toan assay reagent comprising GDNFR protein, wherein said GDNFR proteinreacts with GDNF present in the test sample and produces a detectablereaction product indicative of the presence of GDNF.